Compositions and methods for producing chemicals and derivatives thereof

ABSTRACT

The present invention provides methods for producing a product of one or more enzymatic pathways. The pathways used in the methods of the invention involve one or more conversion steps such as, for example, an enzymatic conversion of guluronic acid into D-glucarate (Step 7); an enzymatic conversion of 5-ketogluconate (5-KGA) into L-Iduronic acid (Step 15); an enzymatic conversion of L-Iduronic acid into Idaric acid Step 7b); and an enzymatic conversion of 5-ketocluconate into 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16). In some embodiments the methods of the invention produce 2,5-furandicarboxylic acid (FDCA) as a product. The methods include both enzymatic and chemical conversions as steps. Various pathways are also provided for converting glucose into 5-dehdyro-4-deoxy-glucarate (DDG), and for converting glucose into 2,5-furandicarboxylic acid (FDCA). The methods also involve the use of engineered enzymes that perform reactions with high specificity and efficiency. Additional products that can be produce include metabolic products such as, but not limited to, guluronic acid, L-iduronic acid, idaric acid, glucaric acid. Any of the products can be produced using glucose as a substrate or using any intermediate in any of the methods or pathways of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.14/033,300, filed Sep. 20, 2013, which claims the benefit of priorityunder 35 U.S.C. §119(e) of U.S. provisional application Ser. No.61/704,408, filed Sep. 21, 2012, which are each hereby incorporated byreference in their entireties, including all tables, figures, andclaims.

INCORPORATION OF SEQUENCE LISTING

The material in the accompanying Sequence Listing is hereby incorporatedby reference into this application. The accompanying sequence listingtext file, name SGI1660-2_txt, was created on Mar. 21, 2014 and is 190KB. The file can be assessed using Microsoft Word on a computer thatuses Windows OS.

BACKGROUND OF THE INVENTION

In recent years, an increasing effort has been devoted to identify newand effective ways to use renewable feedstocks for the production oforganic chemicals. Among a plethora of downstream chemical processingtechnologies, the conversion of biomass-derived sugars to value-addedchemicals is considered very important. In particular, six-carbonedcarbohydrates, i.e. hexoses such as fructose and glucose, are widelyrecognized the most abundant monosaccharides existing in nature,therefore can be suitably and economically used as the chemicalfeedstocks.

The production of furans and furan derivatives from sugars has attractedincreasing attention in chemistry and in catalysis studies, and isbelieved to have the potential to provide one of the major routes toachieving sustainable energy supply and chemicals production. Indeed,dehydration and/or oxidation of the sugars available withinbiorefineries with integrated biomass conversion processes can lead to alarge family of products including a wide range of furans and furanderivatives.

Among the furans having the most commercial values,furan-2,5-dicarboxylic acid (also known as 2,5-furandicarboxylic acid,hereinafter abbreviated as FDCA) is a valuable intermediate with varioususes in several industries including pharmaceuticals, pesticides,antibacterial agents, fragrances, agricultural chemicals, as well as ina wide range of manufacturing applications of polymer materials, e.g.bioplastic resins. As such, FDCA is considered a green alternative ofterephthalic acid (TPA), a petroleum-based monomer that is one of thelargest-volume petrochemicals produced yearly worldwide. In fact, the USDepartment of Energy has identified FDCA as one of the top 12 prioritycompounds made from sugars into a value-added chemical for establishingthe “green” chemistry of the future, and as such, it has been named oneof the “sleeping giants” of the renewable intermediate chemicals (Werpyand Petersen, Top Value Added Chemicals from Biomass. US Department ofEnergy, Biomass, Vol 1, 2004).

Although various methods have been proposed for commercial scaleproduction of FDCA (for review, see, e.g., Tong et al., Appl. CatalysisA: General, 385, 1-13, 2010), the main industrial synthesis of FDCAcurrently relies on a chemical dehydration of hexoses, such as glucoseor fructose, to the intermediate 5-hydroxymethylfurfural (5-HMF),followed by a chemical oxidation to FDCA. However, it has been reportedthat current FDCA production processes via dehydration are generallynonselective, unless immediately upon their formation, the unstableintermediate products can be transformed to more stable materials. Thus,the primary technical barrier in the production and use of FDCA is thedevelopment of an effective and selective dehydration process frombiomass-derived sugars.

It is therefore desirable to develop methods for production of thishighly important compound, as well as many other chemicals andmetabolites, by alternative means that not only would substituterenewable for petroleum-based feedstocks, but also use less energy andcapital-intensive technologies. In particular, the selective control ofsugar dehydration could be a very powerful technology, leading to a widerange of additional, inexpensive building blocks.

SUMMARY OF THE INVENTION

The present invention provides methods for producing a product of one ormore enzymatic pathways. The pathways used in the methods of theinvention involve one or more conversion steps such as, for example, anenzymatic conversion of guluronic acid into D-glucarate (Step 7); anenzymatic conversion of 5-ketogluconate (5-KGA) into L-Iduronic acid(Step 15); an enzymatic conversion of L-Iduronic acid into Idaric acidStep 7b); and an enzymatic conversion of 5-ketogluconate into4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16); an enzymaticconversion of 1,5-gluconolactone to gulurono-lactone (Step 19). In someembodiments the methods of the invention produce 2,5-furandicarboxylicacid (FDCA) as a product. The methods can include both enzymatic andchemical conversions as steps. Various pathways are also provided forconverting glucose or fructose or sucrose or galactose into5-dehydro-4-deoxy-glucarate (DDG), and for converting the same sugarsinto FDCA. The methods can also involve the use of engineered enzymesthat perform reactions with high specificity and efficiency.

In a first aspect the invention provides a method for producing aproduct of an enzymatic or chemical pathway from a starting substrate.The pathway can contain any one or more of the following conversionsteps: an enzymatic conversion of guluronic acid into D-glucarate (Step7); an enzymatic conversion of 5-ketogluconate (5-KGA) into L-Iduronicacid (Step 15); an enzymatic conversion of L-Iduronic acid into Idaricacid (Step 7b); and an enzymatic conversion of 5-ketocluconate into4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16); an enzymaticconversion of 1,5-gluconolactone to gulurono-lactone (Step 19).

In one embodiment the product of the enzymatic pathway is5-dehydro-4-deoxy-glucarate (DDG). In various embodiments the substrateof the method can be glucose, and the product can5-dehydro-4-deoxy-glucarate (DDG). The method can involve the steps ofthe enzymatic conversion of D-glucose to 1,5-gluconolactone (Step 1);the enzymatic conversion of 1,5-gluconolactone to gulurono-lactone (Step19); the enzymatic conversion of gulurono-lactone to guluronic acid(Step 1B); the enzymatic conversion of guluronic acid to D-glucarate(Step 7); and the enzymatic conversion of D-glucarate to5-dehydro-4-deoxy-glucarate (DDG) (Step 8).

In another method of the invention the substrate is glucose and theproduct is DDG, and the method involves the steps of: the conversion ofD-glucose to 1,5-gluconolactone (Step 1); the conversion of1,5-gluconolactone to gluconic acid (Step 1a); the conversion ofgluconic acid to 5-ketogluconate (5-KGA) (Step 14); the conversion of5-ketogluconate (5-KGA) to L-Iduronic acid (Step 15); the conversion ofL-Iduronic acid to Idaric acid (Step 7b); and the conversion of Idaricacid to DDG (Step 8a).

In another method of the invention the substrate is glucose and theproduct is DDG and the method involves the steps of the conversion ofD-glucose to 1,5-gluconolactone (Step 1); the conversion of1,5-gluconolactone to gluconic acid (Step 1a); the conversion ofgluconic acid to 5-ketogluconate (5-KGA) (Step 14); the conversion of5-ketogluconate (5-KGA) to 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH)(Step 16); the conversion of 4,6-dihydroxy 2,5-diketo hexanoate(2,5-DDH) to 4-deoxy-5-threo-hexosulose uronate (DTHU) (Step 4); and theconversion of 4-deoxy-5-threo-hexosulose uronate (DTHU) to DDG (Step 5).

In another method of the invention the substrate is glucose and theproduct is DDG, and the method involves the steps of: the conversion ofD-glucose to 1,5-gluconolactone (Step 1); the conversion of1,5-gluconolactone to gluconic acid (Step 1a); the conversion ofgluconic acid to 5-ketogluconate (5-KGA) (Step 14); the conversion of5-ketogluconate (5-KGA) to L-Iduronic acid (Step 15); the conversion ofL-Iduronic acid to 4-deoxy-5-threo-hexosulose uronate (DTHU) (Step 7B);and the conversion of 4-deoxy-5-threo-hexosulose uronate (DTHU) to DDG(Step 5).

Any of the methods disclosed herein can further involve the step ofconverting the DDG to 2,5-furan-dicarboxylic acid (FDCA). Converting theDDG to FDCA in any of the methods can involve contacting DDG with aninorganic acid to convert the DDG to FDCA.

In another aspect the invention provides a method for synthesizingderivatized (esterified) FDCA. The method involves contacting DDG withan alcohol, an inorganic acid at a temperature in excess of 60 C to formderivatized FDCA. In different embodiments the alcohol is methanol,butanol or ethanol.

In another aspect the invention provides a method for synthesizing aderivative of FDCA. The method involves contacting DDG with an alcohol,an inorganic acid, and a co-solvent to produce a derivative of DDG;optionally purifying the derivative of DDG; and contacting thederivative of DDG with an inorganic acid to produce a derivative ofFDCA. The inorganic acid can be sulfuric acid and the alcohol can beethanol or butanol. In various embodiments the co-solvent can be any ofTHF, acetone, acetonitrile, an ether, butyl acetate, an dioxane,chloroform, methylene chloride, 1,2-dichloroethane, a hexane, toluene,and a xylene.

In one embodiment in the derivative of DDG is di-ethyl DDG and thederivative of FDCA is di-ethyl FDCA, and in another embodiment thederivative of DDG is di-butyl DDG and the derivative of FDCA is di-butylFDCA.

In another aspect the invention provides a method for synthesizing FDCA.The method involves contacting DDG with an inorganic acid in a gasphase.

In another aspect the invention provides a method for synthesizing FDCA.The method involves contacting DDG with an inorganic acid at atemperature in excess of 120 C.

In another aspect the invention provides a method for synthesizing FDCA.The method involves contacting DDG with an inorganic acid underanhydrous reaction conditions.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a electrophoretic gel of crude lysates and purified enzymes ofproteins 474, 475, and 476.

FIGS. 2 a-h is a schematic illustration of the pathways of Routes 1, 2,2A, 2C, 2D, 2E, 2F, respectively.

FIGS. 3 a-c present a schematic illustration of the pathways of Routes3, 4, and 5, respectively.

FIG. 4 is an HPCL-MS analysis of the dehydration of gluconate withgluconate dehydratase to produce DHG by pSGI-359.

FIG. 5 is a graphical illustration of semicarbizide assay plots formeasuring the activity of gluconate dehydratases.

FIGS. 6 a-6 b provide Lineweaver-Burk plots for the oxidation ofglucuronate and iduronate with three enzymes of the invention.

FIG. 7 a shows the results of an HPLC analysis of time points for theisomerization of 5KGA and Iduronate using enzymes DTHU isomerases in theEC 5.3.1.17 family. Controls: dead enzyme is a control with heatinactivated enzyme. Med Bl refers to reactions without isomerase add/n.Time points, x axis 1=0.5 h; 2=1; 3=2 h; 4=16 h. FIG. 7 b shows an HPLCanalysis of time points for the isomerization of 5KGA and iduronateusing enzymes in the EC 5.3.1.17 family. Controls: dead enzyme is acontrol with heat inactivated enzyme; Med Bl: refers to reactionswithout isomerase add/n. Time points, X axis: 1=0 h; 2=1 h; 3=2 h; 4=17h.

FIG. 8 shows product formation for the isomerization of 5KGA andiduronate with enzymes in the EC 5.3.1.n1 family. The data were obtainedfrom enzymatic assays.

FIG. 9: HPLC analysis of the formation of 2,5-DDH and the reduction of5KGA concentration over time. Total ion counts for 2,5-DDH are shown.

FIG. 10 is a HPLC-MS chromatogram showing the production of guluronicacid lactone from 1,5-gluconolactone. An overlay of a trace of authenticguluronic acid is shown.

FIG. 11 is a schematic illustration of the Scheme 6 reaction pathway.

FIGS. 12 a and 12 b are LC-MS chromatograms showing 5-KGA and DDGreaction products, respectively.

FIG. 13 is an LC-MS chromatogram showing FDCA and FDCA dibutyl esterderivative reaction products.

FIG. 14 a is a GC-MS analysis of a crude reaction sample of thediethyl-FDCA synthesis from the reaction of DDG with ethanol. Singlepeak corresponded to diethyl-FDCA. FIG. 14 b is an MS fragmentation ofthe major product from the reaction of DDG with ethanol.

FIG. 15 a is a GC-MS analysis of a crude reaction sample of thediethyl-FDCA synthesis from the reaction of DDG with ethanol. Singlepeak corresponded to diethyl-FDCA. FIG. 15 b is a MS fragmentation ofthe major product from the reaction of DDG with ethanol.

FIG. 16 is a schematic illustration of the synthesis of FDCA and itsderivatives from DTHU.

FIG. 17 is a schematic illustration of Scheme 1. Cell free enzymaticsynthesis of DDG from glucose. Enzymes are ST-1: glucose oxidase; ST-1A:hydrolysis-chemical; ST-14: gluconate dehydrogenase (pSGI-504); ST-15:5-dehydro-4-deoxy-D-glucuronate isomerase (DTHU IS, pSGI-434); ST-7B:Uronate dehydrogenase (UroDH, pSGI-476)); ST-8A Glucarate dehydratase(GlucDH, pSGI-353); ST-A: NAD(P)H oxidase (NADH OX, pSGI-431); ST-B:Catalase. FIG. 17 b shows the concentration of reaction intermediatesover the first 3 h as analyzed by HPLC. Formation of DDG is shown inboth reactions.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for producing a product of anenzymatic pathway. The methods can comprise the enzymatic conversion ofa substrate into a product. By utilizing the enzymatic and chemicalpathways of the invention it is possible to synthesize a wide variety ofproducts in a highly efficient and economical manner. One product thatcan be produced by the methods and pathways of the invention is2,5-furanyl dicarboxylic acid (FDCA), which can be produced atcommercial scales according to the invention. The methods can compriseone or more enzymatic and/or chemical substrate-to-product conversionsteps disclosed herein. In some embodiments the enzymes utilized performenzymatic conversion steps using activities unknown for the enzymes.These novel activities can therefore be employed in the invention toperform the conversion steps and perform a substrate to productconversion as part of a enzymatic and/or chemical pathway. Any of theproducts of any of the pathways disclosed herein (e.g., DDG, iduronicacid, idaric acid, glucaric acid, FDCA, etc.) can be produced on acommercial scale. i.e. in quantities of at least 1 gram or at least 10grams or at least 100 grams or at least 1 kg in a single bioreactor orreaction vessel, as disclosed herein.

The pathways of the invention are comprised of any one or more of thesteps disclosed herein. It is understood that a step of a pathway of theinvention can involve the forward reaction or the reverse reaction,i.e., the substrate A being converted into product B, while in thereverse reaction substrate B is converted into product A. In the methodsboth the forward and the reverse reactions are described as the stepunless otherwise noted.

The methods involve producing a product of a pathway, which can be anenzymatic pathway. The methods involve one or more enzymatic and/orchemical conversion steps, which convert a substrate to a product. Stepsthat can be included in the methods include, for example, any one ormore of: an enzymatic conversion of guluronic acid into D-glucarate(Step 7); an enzymatic conversion of L-Iduronic acid to4-deoxy-5-threo-hexosulose uronate (DTHU) (17); an enzymatic conversionof 5-ketogluconate (5-KGA) into L-Iduronic acid (Step 15); an enzymaticconversion of L-Iduronic acid into Idaric acid Step 7B); and anenzymatic conversion of 5-ketocluconate into 4,6-dihydroxy 2,5-diketohexanoate (2,5-DDH) (Step 16); an enzymatic conversion of1,5-gluconolactone to gulurono-lactone (Step 19). Any one or more of theaforementioned steps can be included in a method or pathway of theinvention. An enzymatic step or pathway is a step or pathway thatrequires an enzyme as a catalyst in the reaction to make the stepproceed. Chemical steps can be performed without an enzyme as a catalystin the reaction. Any one or more of the steps recited in the methods canbe an enzymatic step. In some embodiments every step of the pathway isan enzymatic step, while in other embodiments one or more steps in thepathway is a chemical step.

In some embodiments any of the methods can include a step involving theaddition of the substrate of the reaction to a reaction mix containingthe enzyme that performs the conversion. Thus the method of convertingguluronic acid into D-glucarate (step 7) can involve the addition ofguluronic acid as starting substrate to the reaction mix; the enzymaticconversion of L-iduronic acid to Idaric acid (7B) can involve theaddition of L-Iduronic acid as starting substrate to the reaction mix;the enzymatic conversion of L-Iduronic acid to4-deoxy-5-threo-hexosulose uronate (DTHU) (17) can involve the additionof L-iduronic acid as starting substrate to the reaction mix. Any of themethods can involve a step of adding glucose, fructose, galactose,sucrose, or mannose or another mono- or di-saccharide to the reactionmixture. Another step that can be included in any of the methods is astep of purifying from the reaction mixture a reaction product. Thus, astep of purifying glucaric acid/D-glucarate or L-Iduronicacid/iduronate, or Idaric acid, or 2,5-diketo hexanedioic/DKHA can beincluded in any of the methods described herein. Any of the methodsdisclose can include a step of isolating or purifying DDG or FDCA fromthe reaction mixture. And any of the methods can involve a step ofadding an enzyme that performs any one or more of the steps describedherein to the reaction mixture. A reaction mixture is a mixture of atleast one substrate and at least one enzyme and involves the conversionof at least one substrate into a least one enzyme product. Any of themethods can involve a step of adding an isolated enzyme to a reactionmix, the enzyme performing a substrate to product conversion step of apathway of the invention, and the isolated enzyme being at least 10%purified or at least 20% purified or at least 25% purified or at least50% purified or at least 70% purified or at least 80% purified or atleast 90%, all w/w.

Since many sugars can be converted into other sugars any of the methodsor pathways of the invention can involve the use of glucose, sucrose,fructose or galactose as the starting substrate. Thus, in any pathway orreaction disclosed herein where glucose is the starting substrate it isunderstood that fructose or sucrose or galactose or mannose or anotherstarting substrate can also be a starting substrate for that pathway orreaction. In some embodiments the sugar is converted into glucose whichthen enters the pathway but in other embodiments the pathway begins withfructose or sucrose or galactose or mannose or another mono- ordi-saccharide.

The reactions of the invention can occur in a lysate of cells or acell-free lysate that contains one or more enzymes that perform theenzymatic conversion, but can also occur in a reaction mixturecontaining components added by the user to Rhin a reaction mixture, orcan contain components purified from a cell lysate, or may be containedin a whole cell biocatalyst. The reaction can also occur in a mix madeof purified components that have been combined, such as in a mix wherethe substrate and enzyme were combined to form the reaction mix. Thereactions can occur in an in vitro reaction or can occur in arecombinant cell, and therefore the product(s) can be harvested bylysing the cells or by collecting from the culture medium. The reactionscan occur in a laboratory container or reaction vessel such as, forexample, a centrifuge tube, a test tube, a vial, a beaker, or a glass ormetal or plastic container or reactor, a fermenter or fermentationvessel or bioreactor, an algae pond, any of which can be small scale orlarge scale. Any of the organisms described herein can be utilized ashost cells to produce the product of a step or pathway of the invention.The organisms can also be used to produce one or more enzymes of theinvention for use in a method of the invention. Various types oforganisms can be used. Examples include: bacteria of the familyAcetobacteraceae (e.g. bacteria of the genus Acetobacter, Acidiphilium,Gluconobacter, Gluconoacetobacter), or bacteria of the familyPseudomonadaceae (e.g., genus Azotobacter, Pseudomonas), or bacteria ofthe family Enterobacteriacea (e.g., of the genus Escherichia (e.g., E.coli), Klebsiella). Yeast can also be used for these purposes such asyeast of the genera Saccharomyces, Ashbya, Kluveromyces, Lachancea,Zygosaccharomyces, Candida, Pichia, Arxula or Trichosporon orBlastobotrys. Cyanobacteria can also be used such as those of the genusCyanothece (e.g. Cyanothece strains ATCC 51142, PCC 7424, PCC 7425, PCC7822, PCC 8801, PCC 8802), or Microcystis or Synechococcus (e.g.,strains elongatus PCC 7942, PCC 7002, PCC 6301, CC9311, CC9605, CC9902,JA-2-3B′ a(2-13), JA-3-3Ab, RCC307, WH 7803, WH 8102) or Synechocystis,or Thermosynechococcus. Thus the present invention provides recombinanthost cells comprising a recombinant nucleic acid of one or more of SEQID NOs: 4-6, 20-32, 36-38, 47-54, 56, 62-66, 69-70, 72, and 79-84 or acodon-optimized sequence of any of SEQ ID NOs: 1-84. The host cells canalso contain a vector of the invention described herein. A “codonoptimized” sequence refers to changes in the codons of a sequence tothose preferentially used in a particular organism so that the encodedprotein is efficiently expressed in the organism carrying the sequence.The recombinant nucleic acid sequence can be comprised on a vector, asdisclosed herein.

In various embodiments the methods of the invention are methods ofconverting glucose or fructose or sucrose or galactose to DDG, orglucose or fructose or sucrose or galactose to FDCA, or glucose orfructose or sucrose or galactose to DTHU or DEHU, or for converting DDGto FDCA. The methods can involve converting the starting substrate inthe method into the product. The starting substrate is the chemicalentity considered to begin the method and the product is the chemicalentity considered to be the final end product of the method.Intermediates are those chemical entities that are created in the method(whether transiently or permanently) and that are present in thereaction pathway between the starting substrate and the product. Invarious embodiments the methods and pathways of the invention have aboutfour or about five intermediates or 4-5 intermediates, or about 3intermediates, or 3-5 intermediates, or less than 6 or less than 7 orless than 8 or less than 9 or less than 10 or less than 15 or less than20 intermediates, meaning these values not counting the startingsubstrate or the final end product.

The invention provides methods of producing FDCA and/or DDG, fromglucose or fructose or sucrose or galactose that have high yields. Thetheoretical yield is the amount of product that would be formed if thereaction went to completion under ideal conditions. In differentembodiments the methods of the invention produce DDG from glucose,fructose, or galactose with a theoretical yield of at least 50% molar,or at least 60% molar or at least 70% molar, or at least 80% molar, atleast 90% molar or at least 95% molar or at least 97% molar or at least98% molar or at least 99% molar, or a theoretical yield of 100% molar.The methods of the invention also can provide product with a carbonconservation of at least 80% or at least 90% or at least 95% or at least97% or at least 98% or at least 99% or 100%, meaning that the particularcarbon atoms present in the initial substrate are present in the endproduct of the method at the recited percentage. In some embodiments themethods produce DDG and/or FDCA from glucose or fructose or sucrose orgalactose via dehydration reactions.

Example Synthesis Routes

The invention also provides specific pathways for synthesizing andproducing a desired product. Any of the following described routes orpathways can begin with glucose or fructose or sucrose or galactose ormannose and flow towards a desired product. In some embodimentsD-glucose is the starting substrate and the direction of the pathwaytowards any intermediate or final product of the pathway is consideredto be in the downstream direction, while the opposite direction towardsglucose is considered the upstream direction. It will be realized thatroutes or pathways can flow in either the downstream or upstreamdirection. While glucose is used as an example starting substrate forpathways described herein, it is also understood that sucrose, fructose,galactose, or mannose or any intermediate in any of the pathways canalso be the starting substrate in any method of the invention, and DDG,DTHU, FDCA, or any intermediate in any of the routes or pathways of theinvention can be the final end product of a method of the invention. Thedisclosed methods therefore include any one or more steps disclosed inany of the routes or pathways of the invention for converting anystarting substrate or intermediate into any end product or intermediatein the disclosed routes or pathways using one or more of the steps inthe disclosed routes or pathways. Thus, for example the methods can bemethods for converting glucose or fructose or sucrose or galactose ormannose to DDG, or to guluronic acid, or to galactarate, or to DTHU, orto DEHU, or to guluronic acid, or to iduronic acid, or to idaric acid,or to glucaric acid, or for converting galactarate to DDG, or forconverting guluronic acid to D-glucarate, or for converting 5-KGA toL-Iduronic acid, or for converting L-Iduronic acid to Idaric acid, orfor converting 5-KGA to 2,5-DDH or DTHU, or for converting DHG to DEHU.In these embodiments the methods utilize the steps disclosed in themethods and pathways of the invention from starting substrate to therelevant end product. One or more of the steps can also be utilized inmethods flowing in the “opposite” or upstream direction from thepathways disclosed herein.

Route 1 is illustrated in FIG. 2 a. Route 1 converts D-glucose (or anyintermediate in the pathway) into 5-dehydro-4-deoxy-glucarate (DDG) viaan enzymatic pathway via a series of indicated steps. Route 1 convertsD-glucose into DDG via a pathway having 1,5-gluconolactone, gluconicacid, 3-dehydro-gluconic acid (DHG), 4,6-dihydroxy 2,5-diketo hexanoate(2,5-DDH), and 4-deoxy-L-threo-hexosulose uronate (DTHU) asintermediates and DDG as the final end product. For any of the pathwaysadditional intermediates not shown can also be present. The steps arethe enzymatic conversion of D-glucose to 1,5-gluconolactone (Step 1);the enzymatic conversion of 1,5-gluconolactone to gluconic acid (Step1A); the enzymatic conversion of gluconic acid to 3-dehydro-gluconicacid (DHG) (Step 2); the enzymatic conversion of 3-dehydro-gluconic acid(DHG) to 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 3); theenzymatic conversion of 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) to4-deoxy-L-threo-hexosulose uronate (DTHU) (Step 4); and the enzymaticconversion of 4-deoxy-L-threo-hexosulose uronate (DTHU) to5-dehydro-4-deoxy glucarate (DDG) (Step 5). Route 1 also comprisessub-routes where the glucose or any intermediate in the pathway as asubstrate is converted into any other downstream intermediate as finalproduct, and each substrate to product sub-route is considered disclosedas if each is set forth herein in full.

Route 2 is illustrated in FIG. 2 b and converts D-glucose into DDG. Thesteps in the Route 2 pathway are the enzymatic conversion of D-glucoseinto 1,5-gluconolactone (Step 1); the enzymatic conversion of1,5-gluconolactone to gluconic acid (Step 1A); the enzymatic conversionof gluconic acid to guluronic acid (Step 6); the enzymatic conversion ofguluronic acid to D-glucarate (Step 7); the enzymatic conversion ofD-glucarate to DDG (Step 8). Route 2 also comprises sub-routes whereglucose or any intermediate in the pathway as substrate is convertedinto any other downstream intermediate as final product, and eachsub-route is considered disclosed as if each is set forth herein infull. For example in some embodiments the methods comprise steps for theconversion of glucose or gluconic acid as substrate into guluronic acidor D-glucarate as product using one or more of the steps described inRoute 2.

Route 2A is illustrated in FIG. 2 c. The steps in Route 2A are theenzymatic conversion of D-glucose to 1,5-gluconolactone (Step 1); theenzymatic conversion of 1,5-gluconolactone to guluronic acid lactone(Step 19); the enzymatic conversion of guluronic acid lactone toguluronic acid (Step 1B); the enzymatic conversion of guluronic acid toD-glucarate (Step 7); the enzymatic conversion of D-glucarate to5-dehydro-4-deoxy-glucarate (DDG) (Step 8). Route 2A also comprisessub-routes where glucose or any intermediate in the pathway as startingsubstrate is converted into any other downstream intermediate as finalend product, and each sub-route is considered disclosed as if each isset forth herein in full. For example in some embodiments the methodscomprise steps for the conversion of glucose or guluronic acid lactoneas substrate into glucarate or DDG as product using one or more of thesteps described in Route 2A.

Route 2B is illustrated in FIG. 2 d. The steps in Route 2B are theenzymatic conversion of D-glucose into gluconic acid (Steps 1 and 1A);the enzymatic conversion of gluconic acid into 5-ketogluconate (5-KGA)(Step 14); the enzymatic conversion of 5-KGA into L-Iduronic acid (Step15); the enzymatic conversion of L-Iduronic acid into Idaric acid (Step7B); the enzymatic conversion of Idaric acid into DDG (Step 8A). Route2B also comprises sub-routes where glucose or any intermediate in thepathway as starting substrate is converted into any other downstreamintermediate as final end product, and each sub-route is considereddisclosed as if each is set forth herein in full. For example in someembodiments the methods comprise steps for the conversion of glucose or5-KGA as substrate into iduronic acid or idaric acid as product usingone or more of the steps described in Route 2B.

Route 2C is illustrated in FIG. 2 e. The steps in Route 2C are theenzymatic conversion of D-glucose to gluconic acid (Steps 1 and 1A); theenzymatic conversion of gluconic acid to 5-ketogluconate (5-KGA) (Step14); the enzymatic conversion of 5-KGA to 4,6-dihydroxy 2,5-diketohexanoate (2,5-DDH) (Step 16); the enzymatic conversion of 4,6-dihydroxy2,5-diketo hexanoate (2,5-DDH) to 4-deoxy-5-threo-hexosulose uronate(DTHU) (Step 4); the enzymatic conversion of DTHU to DDG (Step 5). Route2C also comprises sub-routes where glucose or any intermediate in thepathway as starting substrate is converted into any other downstreamintermediate as final end product, and each sub-route is considereddisclosed as if each is set forth herein in full. For example in someembodiments the methods comprise steps for the conversion of glucose orgluconic acid as substrate into 2,5-DDH or DTHU using one or more stepsdescribed in Route 2C.

Route 2D is illustrated in FIG. 2 f. The steps in Route 2D are theenzymatic conversion of D-glucose to gluconic acid (Steps 1 and 1A); theenzymatic conversion of gluconic acid to 5-ketogluconate (5-KGA) (Step14); the enzymatic conversion of 5-KGA to Iduronic acid (Step 15); theenzymatic conversion of L-Iduronic acid to DTHU (Step 17); the enzymaticconversion of DTHU to DDG (Step 5). Route 2D also comprises sub-routeswhere glucose or any intermediate in the pathway as starting substrateis converted into any other downstream intermediate as final endproduct, and each sub-route is considered disclosed as if each is setforth herein in full. For example in some embodiments the methodscomprise steps for the conversion of glucose or 5-KGA as substrate intoL-iduronic acid or DTHU using one or more of the steps described inRoute 2D.

Route 2E is illustrated in FIG. 2 g. The steps in Route 2D are theenzymatic conversion of D-glucose to 1,5-gluconolactone (Step 1); theenzymatic conversion of 1,5-gluconolactone to guluronic acid lactone(Step 19); the enzymatic conversion of guluronic acid lactone toguluronic acid (Step 1B); the enzymatic conversion of guluronic acid to4-deoxy-erythro-hexosulose uronate (DEHU) (Step 17A); the enzymaticconversion of DEHU to 3-deoxy-D-erythro-2-hexylosaric acid (DDH) (Step7A). Route 2E also comprises sub-routes where glucose or anyintermediate in the pathway as starting substrate is converted into anyother downstream intermediate as final end product, and each sub-routeis considered disclosed as if each is set forth herein in full. Forexample in some embodiments the methods comprise steps for theconversion of glucose as substrate into guluronic acid or DEHU using oneor more of the steps described in Route 2E.

Route 2F is illustrated in FIG. 2 h. The steps in Route 2F are theenzymatic conversion of D-glucose to gluconic acid (Steps 1 and 1A); theenzymatic conversion of gluconic acid to guluronic acid (Step 6); theenzymatic conversion of guluronic acid to 4-deoxy-erythro-hexosuloseuronate (DEHU) (Step 17A); the enzymatic conversion of DEHU to3-deoxy-D-erythro-2-hexylosaric acid (DDH) (Step 7A). Route 2F alsocomprises sub-routes where glucose or gluconic acid or any intermediatein the pathway as starting substrate is converted into guluronic acid orDDH or any other downstream intermediate as final end product using oneor more of the steps of Route 2F, and each sub-route is considereddisclosed as if each is set forth herein in full.

Route 3 is illustrated in FIG. 3 a. The steps in Route 3 are theenzymatic conversion of D-glucose to gluconic acid (Steps 1 and 1A); theenzymatic conversion of gluconic acid to 3-dehydro-gluconic acid (DHG)(Step 2); the enzymatic conversion of DHG to 4-deoxy-erythro-hexosuloseuronate (DEHU) (Step 6A); the enzymatic conversion of DEHU to DDG (Step7A). Route 3 also comprises sub-routes where glucose or fructose orsucrose or galactose or any intermediate in the pathway as startingsubstrate is converted into gluconic acid or DDH any other downstreamintermediate of Route 3 as final end product using one or more of thesteps of Route 3, and each sub-route is considered disclosed as if eachis set forth herein in full.

Route 4 is illustrated in FIG. 3 b. The steps in Route 4 are theenzymatic conversion of D-glucose to a-D-gluco-hexodialdo-1,5-pyranose(Step 9); the enzymatic conversion of a-D-gluco-hexodialdo-1,5-pyranoseto a-D-glucopyranuronic acid (Step 10); the enzymatic conversion ofa-D-glucopyranuronic acid to D-glucaric acid 1,5-lactone (Step 11); theenzymatic conversion of D-glucaric acid 1,5-lactone to D-glucarate (Step1C); the enzymatic conversion of D-glucarate to DDG (Step 8). Route 4also comprises sub-routes where glucose or any intermediate in thepathway as starting substrate is converted into glucarate or DDG or anyother downstream intermediate as final end product using one or more ofthe steps of Route 4, and each sub-route is considered disclosed as ifeach is set forth herein in full.

Route 5 is illustrated in FIG. 3 c. The steps in Route 5 are theenzymatic conversion of D-galactose to D-galacto-hexodialdose (Step 9A);the enzymatic conversion of D-galacto-hexodialdose to galacturonate(Step 10A); the enzymatic conversion of galacturonate to galactarate(Step 11A); the enzymatic conversion of galactarate to DDG (Step 13).Route 5 also comprises sub-routes where galactose or any intermediate inthe pathway as starting substrate is converted into any other downstreamintermediate as final product, and each sub-route is considereddisclosed as if each is set forth herein in full. For example in someembodiments the methods comprise steps for the conversion of galactoseor another substrate into galacturonate or galactarate using the stepsdescribed in Route 5.

In various other embodiments the invention provides a method ofproducing a product of an enzymatic and/or chemical pathway from astarting substrate that involves performing Step 1, followed by Step 19,followed by Step 1B to produce a guluronic acid product. Optionally thepathway can continue with Step 7 to produce glucarate. In anotherembodiment the method involves performing Steps 1 and 1A followed byStep 14, followed by Step 15 to produce Iduronic acid. Optionally themethod can continue with Step 7B to produce an Idaric acid product orwith Step 17 to produce DTHU. In another embodiment the method involvesperforming Steps 1 and 1A, followed by Step 14 followed by Step 16 toproduce a 2,5-DDH product. In another embodiment the method involvesperforming Step 1 followed by Step 19 to produce guluronic acid lactone.

The Enzymatic Steps

There are disclosed a wide variety of enzymes (and nucleic acids thatencode the enzymes) that can perform the steps of the methods outlinedherein. The enzymes utilized in the enzymatic steps of the invention canbe proteins or polypeptides. In addition to the families and classes ofenzymes disclosed herein for performing the steps of the invention,homologs having a sequence identity to any enzyme or nucleic acid or toany of SEQ ID NOs 1-84, disclosed herein will also be useful in theinvention. Enzymes and nucleic acids that are homologs of SEQ ID NOs:1-84 have a sequence identity of at least 40% or at least 50% or atleast 60% or at least 70% or at least 80% or at least 90% or at least95% or at least 97% or at least 98% or at least 99% to any nucleic acidor enzyme of SEQ ID NO: 1-84, or to a member of an enzyme classdisclosed herein. Percent sequence identity or homology with respect toamino acid or nucleotide sequences is defined herein as the percentageof amino acid or nucleotide residues in the candidate sequence that areidentical with the known polypeptides, after aligning the sequences formaximum percent identity and introducing gaps, if necessary, to achievethe maximum percent identity or homology. Homology or identity at thenucleotide or amino acid sequence level may be determined using methodsknown in the art, including but not limited to BLAST (Basic LocalAlignment Search Tool) analysis using the algorithms employed by theprograms blastp, blastn, blastx, tblastn and tblastx (Altschul (1997),Nucleic Acids Res. 25, 33 89-3402, and Karlin (1990), Proc. Natl. Acad.Sci. USA 87, 2264-2268), which are tailored for sequence similaritysearching. Alternatively a functional fragment of any of the enzymes ornucleic acids encoding such enzymes or of any enzyme or nucleic acid ofSEQ ID NOs 1-84 disclosed herein may also be used. The term “functionalfragment” refers to a polypeptide that has an amino-terminal and/orcarboxy-terminal deletion and/or internal deletion (which can bereplaced to form a chimeric protein), where the remaining amino acidsequence has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity to the corresponding positions in the referencesequence, and/or that retains about 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the full-lengthpolypeptide. The EC numbers provided use the enzyme nomenclature of theNomenclature Committee of the International Union of Biochemistry andMolecular Biology. In other embodiments the functional fragment retainsthe requirement of the presence of a co-factor necessary for theactivity of a protein or protein encoded by SEQ ID NO:1-84.

Also disclosed is an expression vector having a sequence of SEQ ID NO:4-6, 20-32, 36-38, 47-54, 56, 62-66, 69-70, 72, and 79-84. The vectorcan be a bacterial, yeast, or algal vector. Vectors designed forexpression of a gene can also include a promoter active in the organismcarrying the vector and operably linked to the sequence of theinvention. The vector can contain a promoter or expression controlsequence operatively linked to a sequence of SEQ ID NOs: 4-6, 20-32,36-38, 47-54, 56, 62-66, 69-70, 72, and 79-84 or a codon-optimizedsequence of any of them. A “promoter” refers to a nucleic acid sequencecapable of binding RNA polymerase to initiate transcription of a gene ina 5′ to 3′ (“downstream”) direction. A sequence is “operably linked” toa promoter when the binding of RNA polymerase to the promoter is theproximate cause of said gene's transcription.

Step 1—Conversion (oxidation or dehydrogenation) of glucose to1,5-gluconolactone. This step can be performed with various enzymes,such as those of the family oxygen dependent glucose oxidases (EC1.1.3.4) or NAD(P)-dependent glucose dehydrogenases (EC 1.1.1.118, EC1.1.1.119). Gluconobacter oxydans has been shown to efficiently oxidizeglucose to gluconic acid and 5-ketogluconate (5-KGA) when grown in afermentor. Enzymes of the family of soluble and membrane-boundPQQ-dependent enzymes (EC 1.1.99.35 and EC 1.1.5.2) found inGluconobacter and other oxidative bacteria can be used. Quinoproteinglucose is another enzyme that is useful in performing this step. Thespecific enzyme selected will be dependent on the desired reactionconditions and necessary co-factors that will be present in thereaction, which are illustrated in Table 1.

Step 1A—Conversion (e.g., hydrolysis) of 1,5-gluconolactone togluconate. This step can be performed chemically in aqueous media andthe rate of hydrolysis is dependent on pH (Shimahara, K, Takahashi, T.,Biochim. Biophys. Acta (1970), 201, 410). Hydrolysis is faster in basicpH (e.g. pH 7.5) and slower in acid pH. Many microorganisms also containspecific 1,5-glucono lactone hydrolases, and a few of them have beencloned and characterized (EC 3.1.1.17; Shinagawa, E Biosci. Biotechnol.Biochem. 2009, 73, 241-244).

Step 1B—Conversion of Guluronic acid lactone to guluronic acid. Thechemical hydrolysis of guluronic acid lactone can be done by aspontaneous reaction in aqueous solutions. An enzyme capable ofcatalyzing this hydrolysis is identified amongst the large number oflactonases (EC 3.1.1. XX and more specifically 3.1.1.17, 3.1.1.25).

Step 2—Conversion of gluconic acid to 3-dehydro gluconic acid (DHG):Several enzymes, such as gluconate dehydratases, can be used in thedehydration of gluconic acid to dehydro gluconic acid (DHG). Examplesinclude those belonging to the gluconate dehydratase family (EC4.2.1.39). A specific example of such a dehydratase has been shown todehydrate gluconate (Kim, S. Lee, S. B. Biotechnol. Bioprocess Eng.(2008), 13, 436). Particular examples of enzymes from this family andtheir cloning are shown in Example 1.

Step 3: Conversion of 3-dehydro-gluconic acid (DHG) to 4,6-dihydroxy2,5-diketo hexanoate (2,5-DDH). Enzymes, 2-dehydro-3-deoxy-D-gluconate5-dehydrogenase (or DHG dehydrogenases) (EC 1.1.1.127) for performingthis conversion have been described.

Step 4: Conversion of 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) to4-deoxy-L-threo-hexosulose uronate (DTHU). Enzymes of the family EC5.3.1.12 can be used in this step, and Step 15 shows that five suchenzymes were cloned and shown to have activity for the dehydration of5-KGA. These enzymes will also show activity towards 2,5-DDH and DTHU.

Step 5: Conversion of DTHU to 5-dehydro-4-deoxy-glucarate (DDG). DDG canbe produced from the chemical or enzymatic oxidation of DTHU, forexample with a mild chemical catalyst capable of oxidizing aldehydes inthe presence of alcohols. Aldehyde oxidases can be used to catalyze thisoxidation. Oxidative bacteria such as Acetobacter and Gluconobacter(Hollmann et al Green Chem. 2011, 13, 226) will be useful in screening.Enzymes of the following families can perform this reaction: aldehydeoxidase EC1.2.3.1, aldehyde ferredoxin oxidoreductase (EC1.2.7.5), andin all the families of EC1.2.1.-XX. Enzymes of the family of uronatedehydrogenases (EC 1.1.1.203) (e.g. see Step 7) will also have thisactivity. Other enzymes with both alcohol and aldehyde oxidationactivity can be used, including enzymes in the alditol oxidase family(see Steps 19 and 6). Other broad substrate oxidases include soluble andmembrane bound PQQ-dependent alcohol/aldehyde oxidases. Morespecifically soluble periplasmic PQQ oxidases enzymes and their homologsbelonging into Type I (EC 1.1.9.1) and II (EC 1.1.2.8) families as wellas membrane bound PQQ oxidases belonging into EC 1.1.5.X families areuseful. In other embodiments aldehyde dehydrogenases/oxidases that acton DTHU can be used.

Step 5 can also be performed using a dehydrogenase from acetic acidbacteria such Gluconobacter and Acetobacter and Gluconoacetobacter, andothers. Whole cell activity is identified by screening microorganismsfor the oxidation of DTHU. The activity is identified and one or more ofthe enzymes is cloned. Enzymes with uronate dehydrogenase activitydescribed in Step-7 and 7B are also screened and found to have thisactivity. A library of soluble periplasmic and membrane boundPQQ-dependent enzymes is also cloned and several enzymes are foundhaving this activity. Some of the enzymes found to have the activity areNAD(P)- or PQQ-dependent dehydrogenases, but others are FAD-dependentaldehyde dehydrogenases. SEQ ID NO: 71-72 are examples of NADP-dependentdehydrogenases, and any one or a combination of them can be used toperform Step 5. SEQ ID NOs: 73-84 are examples of suitable PQQ-dependentdehydrogenases and any one or any combination of them can be used toperform Step 5.

Steps 6 and 6A: Conversion of gluconic acid to guluronic acid (6) andconversion of 3-dehydro-gluconic acid (DHG) to4-deoxy-5-erythro-hexosulose uronate (DEHU) (6A). The enzymes describedin Step 5 are useful for these conversions. Other useful enzymes includeNAD(P)-dependent dehydrogenases in the EC 1.1.1.XX families and morespecifically glucuronate dehydrogenase (EC 1.1.1.19), glucuronolactonereductase (EC 1.1.1.20). In addition, a large number O₂-dependentalcohol oxidases with broad substrate range including sugars will beuseful (EC 1.1.3.XX), including sorbitol/mannitol oxidases (EC1.1.3.40), hexose oxidases (EC 1.1.3.5), alcohol oxidases (EC 1.1.3.13)and vanillin oxidase (EC 1.1.3.38). PQQ-dependent enzymes and enzymespresent in oxidative bacteria can also be used for these conversions.

Steps 7 and 7B: Conversion of guluronic acid to D-glucaric acid (7) andconversion of L-Iduronic acid to Idaric acid (7B). These steps can beaccomplished with enzymes of the family of uronate dehydrogenases (EC1.1.1.203) or the oxidases, as described herein. Examples of uronatedehydrogenases include SEQ ID NO: 1-6, and any one or any combination ofthem can be used to perform Steps 7 and 7B.

Step 7A: Conversion of 4-deoxy-5-erythro-hexosulose uronate (DEHU) to3-deoxy-D-erythro-2-hexylosaric acid (DDH). The same enzymes describedin Step 5 will be useful for performing this conversion. Similar to Step5, for steps 7 and 7B enzymes are identified having the stated activity,which are NAD(P)- or PQQ-dependent dehydrogenases, but others areFAD-dependent aldehyde dehydrogenases. Examples of NADP-dependentgluconate-5-dehydrogenases include SEQ NO: 71-72 and examples ofPQQ-dependent dehydrogenases include SEQ ID NO: 73-84, and any one orany combination of them can be used to perform steps 7 and 7B.

Steps 8 and 8A: Conversion of D-glucaric acid to5-dehydro-4-deoxy-glucarate (DDG) (Step 8) and conversion of Idaric acidto DDG (Step 8A). Enzymes of the family of glucarate dehydratases (EC4.2.1.40) can be used to perform these steps. Enzymes of this familyhave been cloned and have been shown to efficiently convert glucarate toDDG. Two D-glucarate dehydratases (EC 4.2.1.40) were cloned as shown inthe Table of cloned glucarate dehydratases below. Both enzymes showedvery high activity for the dehydration of Glucarate to DDG using thesemicarbazide assay, as described in Step 2.

Cloned glucarate dehydratases Organism pSGI (Vector) Gene ID WT/SYN E.coli 353 (pET28) P0AES2 WT Pseudomonas (SGI) 244 #8114 WT

Step 9 and 9A: Conversion of D-glucose toα-D-gluco-hexodialdo-1,5-pyranose (9) and conversion of D-galactose toD-galacto-hexodialdose (9A). Oxidases such as those of the galactoseoxidase family (EC 1.1.3.9) can be used in this step. Mutant galactoseoxidases are also engineered to have activity on glucose and have beendescribed (Arnold, F. H. et al ChemBioChem, 2002, 3(2), 781). Step 9Acan be performed with enzymes of the class EC 1.1.3.9.

Step 10: Conversion of α-D-gluco-hexodialdo-1,5-pyranose toα-D-glucopyranuronic acid (step 10) and D-galacto-hexodialdose togalacturonate (10A). This step can be performed using an enzyme of thefamily of aldehyde dehydrogenases. Also an enzyme identified from thoseof Step 5 will be useful for both of these conversions.

Step 11 and 11A: Conversion of α-D-glucopyranuronic acid to glucuronicacid 1,5-lactone. Aldehyde dehydrogenases and oxidases as described inStep 5 will be useful in performing this step. Uronate dehydrogenasesdescribed in Steps 7 and 7B can also be useful in performing this step.Step-11A is the conversion of galacturonate to galactarate. The uronatedehydrogenase (EC 1.1.1.203), for example those described in Steps 7 and7B, will be useful in performing this step.

Step 12: Conversion of fructose to glucose. Glucose and fructoseisomerases (EC 5.3.1.5) will be useful in performing this step.

Step 13: Conversion of galactarate to 5-dehydro-4-deoxy-D-glucarate(DDG). Enzymes of the family of galactarate dehydrogenases (EC 4.2.1.42)can be used to perform this step, and additional enzymes can beengineered for performing this step.

Step 14: Conversion of gluconate to 5-ketogluconate (5-KGA). A number ofenzymes of the family of NAD(P)-dependent dehydrogenases (EC1.1.1.69)have been cloned and shown to have activity for the oxidation ofgluconate or the reduction of 5KGA. For example, the NADPH-dependentgluconate 5-dehydrogenase from Gluconobacter (Expasy P50199) wassynthesized for optimal expression in E. coli as shown herein and wascloned in pET24 (pSGI-383). The enzyme was expressed and shown to havethe required activities. Additional enzymes useful for performing thisstep include those of the family of PQQ-dependent enzymes present inGluconobacter (Peters, B. et al. Appl. Microbiol Biotechnol., (2013),97, 6397), as well as the enzymes described in Step 6. Enzymes fromthese families can also be used to synthesize 5KGA from gluconate.

Step 15: Conversion of 5-KGA to L-Iduronic acid. This step can beperformed with various enzymes from different isomerase families, asfurther described in Example 4. Examples include isomerases of SEQ IDNOs: 7-19 or a homolog having at least 70% sequence identity to anisomerase of SEQ ID NOs: 7-19; or by an isomerase encoded by a nucleicacid of SEQ ID NOs: 20-32 or a homolog of any of them.

Step 16: Conversion of 5-KGA to (4S)-4,6-dihydroxy 2,5-diketo hexanoate(2,5-DDH). This dehydration can be performed with enzymes in thegluconate dehydratase family (EC 4.2.3.39), such as those described inExample 5 or Step 17. Examples of gluconate dehydratases that can beused for Step 16 include SEQ ID NOs 33-35 (encoded by SEQ ID NOs: 36-38,and any one or any combination of them can be used to perform Step 16,or homologs thereof.

Step 17 and 17A: L-Iduronate to 4-deoxy-5-threo-hexosulose uronate(DTHU) and Guluronate to 4-deoxy-erythro-5-hexosulose uronate (DEHU).

Enzymes of the family of dehydratases are identified that can be used inthe performance of this step. Enzymes from the families of gluconate orglucarate dehydratases will have the desired activity for performingthese steps. Furthermore, many dehydratases of the family (EC 4.2.1.X)will be useful in the performance of these steps. In particular, enzymesthat dehydrate 1,2-dyhydroxy acids to selectively produce 2-keto-acidswill be useful, such as enzymes of the families: EC 4.2.1.6 (galactonatedehydratase), EC 4.2.1.8 (mannonate dehydratase), EC 4.2.1.25 (arabonatedehydratase), EC 4.2.1.39 (gluconate dehydratase), EC 4.2.1.40(glucarate dehydratase), EC 4.2.1.67 (fuconate dehydratase), EC 4.2.1.82(xylonate dehydratase), EC 4.2.1.90 (rhamnonate dehydratase) anddihydroxy acid dehydratases (4.2.1.9). Since known enzyme selectivity isthe production of an alpha-keto acid the identified enzymes will produceDEHU and DTHU, respectively, as the reaction products Step 19:Conversion of 1,5-gluconolactone to guluronic acid lactone. This stepcan be performed by enzymes of the family of alditol oxidases (EC1.1.3.41) or the enzymes described in Step 6. Examples of alditoloxidases that can be used for Step 19 include SEQ ID NOs 39-54 or ahomolog of any of them, or by an alditol oxidase encoded by a nucleicacid of SEQ ID NOs: 47-54 or a homolog of any of them; and any one orany combination of them can be used to perform Step 19. Methods ofConverting DDG to FDCA and of making esterified DDG and FDCA The presentinvention also provides novel methods of converting DDG to FDCA and FDCAesters. Esters of FDCA include diethyl esters, dibutyl esters, and otheresters. The methods involve converting DDG into a DDG ester bycontacting DDG with an alcohol, an inorganic acid, and optionally aco-solvent to produce a derivative of DDG. The alcohol can be methanol,ethanol, propanol, isopropanol, butanol, isobutanol, pentanol, hexanol,heptanol, octanol, nonanol, decanol, undecanol, dodecanol, tridecanol,tetradecanol, pentadecanol, hexadecanol, heptadecanol, octadecanol,nonadecanol, eicosanol, dimethyl sulfoxide, dimethylformamide,polyethylene glycol, methyl isobutyl ketone, or any C1-C20 alcohol. Theinorganic acid can be sulfuric acid, phosphoric acid, perchloric acid,nitric acid, hydrochloric acid, hydrofluoric acid, hydroboromic acid andhydriodic acid. The co-solvent can be any of or any mixture of THF,acetone, acetonitrile, an ether, butyl acetate, an dioxane, chloroform,methylene chloride, 1,2-dichloroethane, a hexane, toluene, and a xylene.Any combination of the alcohols, inorganic acids, and co-solvents can beutilized in the reactions. The esterified DDG can then be converted intoesterified FDCA, for example by contacting it with an acid catalyst.

DDG Purification

DDG purification for dehydration or esterification was performed byacidifying the DDG, e.g., by lowering the pH of the reaction with theaddition of conc HCl to pH˜2.5. At this pH proteins and any residualglucarate precipitate are removed by filtration and the mixture islyophilized to give a white powder consisting of DDG and the reactionsalts. The mixture can be lyophilized at neutral pH after the enzymeshave been removed by filtration. Without further purification the DDGcan then be dehydrated to give 2,5-FDCA, or be esterified to dibutyl-DDG(or di-ethyl DDG) prior to dehydration. One or more steps of purifyingor esterifying DDG can be added to any of the methods and pathwaysdisclosed herein that produce DDG. Other methods for purifying DDG fromthe aqueous mixture can also be used. These include separations usingmembranes or ion exchange resins that capture salts or DDG etc.

The invention therefore provides a method of purifying DDG that involvesacidifying DDG in a solution, filtering the solution through a filtermembrane, and removing water from the solution (e.g., by lyophilizationro spray drying). The solution with the DDG can be acidified to a pH of2.5-3.5 or pH of 3.0-4.0 or pH of 3.5-4.5 or pH of 4.0-5.0 or pH of4.5-5.5 or pH of 5.0-6.0 or pH of 5.5-6.5 or pH of 6.0-7.0 or pH of6.5-7.5 or pH of 7.0-8.0 or pH of 7.5-8.5 or pH of about 8. The amountof water removed can be greater than 80% or greater than 85% or greaterthan 87% of the water or greater than 90% of the water or greater than95% of the water or greater than 97% or greater than 98% or greater than99% of the water from the solvent comprising the DDG. Yields of greaterthan 25% or 30% or 35% or 40% or 45% molar can be obtained. In oneembodiment the method does not involve a step of ion exchangechromatography.

Methods for Synthesizing FDCA and FDCA Derivatives

The invention also provides various methods of synthesizing FDCA. Onemethod for synthesizing FDCA involves contacting DDG with an alcohol, aninorganic acid at a high temperature to form FDCA. The alcohol can beany alcohol (e.g., any of those described above), and examples include(but are not limited to) methanol, ethanol, propanol, and butanol. Diolscan also be used. The high temperature can be a temperature greater than70° C. or greater than 80° C. or greater than 90° C. or greater than100° C. or greater than 110° C. or greater than 120° C. or greater than130° C. or greater than 140° C. or greater than 150° C. to form FDCA.Reaction yields of greater than 20% or greater than 30% or greater than35% or greater than 40% can be achieved.

The invention also provides methods for synthesizing derivatives ofFDCA. The methods involve contacting a derivative of DDG with aninorganic acid to produce a derivative of FDCA. The inorganic acid canbe, for example, sulfuric acid, or any inorganic acid such as thosedescribed above. Optionally, the derivative of DDG can be purified priorto contacting it with the second inorganic acid. Non-limiting examplesof derivatives of DDG or FDCA include, but are not limited to, methylDDG, ethyl DDG, propyl DDG, butyl DDG, isobutyl DDG, di-methyl DDG,di-ethyl DDG, di-propyl DDG, di-butyl DDG. The derivative of FDCAproduced can be, but is not limited to, methyl FDCA, ethyl FDCA, propylFDCA, butyl FDCA, di-methyl FDCA, di-ethyl FDCA, di-propyl FDCA,di-butyl FDCA, and isobutyl FDCA. The derivate of FDCA producedcorresponds to the derivative of DDG used in the method. The derivativeof FDCA can then be de-esterified to produce FDCA. The method can alsobe conducted in the gas phase, e.g., using the parameters describedbelow.

Another method for synthesizing FDCA or derivatives of FDCA involvescontacting DDG or derivatives of DDG (any described herein) with aninorganic acid in a gas phase, which can be done with a short residencetime, e.g., of less than 10 seconds or less than 8 seconds, or less than6 seconds or less than 5 seconds or less than 4 seconds or less than 3seconds or less than 2 seconds or less than 1 second. The residence timerefers to the time that the sample is present in the reaction zone ofthe high temperature flow through reactor. The method can also beconducted at high temperatures, for example at temperatures greater than150° C., greater than 200° C., greater than 250° C., greater than 300°C. or greater than 350° C. Yields of greater than 25% or greater than30% or greater than 40% or greater than 45% or greater than 50% molarare obtainable. Another method for synthesizing FDCA involves contactingDDG with an inorganic acid at a temperature in excess of 80° C. or 90°C. or 100° C. or 110° C. or 120° C. Another method for synthesizing FDCAinvolves contacting DDG with an inorganic acid under anhydrous reactionconditions. In various embodiments the anhydrous conditions can beestablished by lyophilizing the DDG in any method of synthesizing FDCAdisclosed herein so that the DDG contains less than 10% or less than 9%or less than 8% or less than 7% or less than 6% or less than 5% or lessthan 4% or less than 3% water or less than 2% water, by weight.

The methods of the invention for synthesizing FDCA and its derivativesas described herein provide a significantly higher yield than has beenavailable. In different embodiments molar yields of FDCA (v. DDG) can beobtained of greater than 10% or greater than 15% or greater than 20% orgreater than 25% or greater than 30% or greater than 35% or greater than40% or greater than 45% or greater than 50% or greater than 60% orgreater than 65% or from about 40% to about 70%, or from about 45% toabout 65%, or from about 50% to about 60%.

EXAMPLES Example 1 Step 2, Gluconic Acid to 3-dehydro-gluconic acid(DHG)

Enzymes with natural activity for the dehydration of gluconate areuseful in the invention (EC 4.2.1.39). Three enzymes from this familywere cloned as shown in Table 1. Enzyme pSGI-365 was cloned and shown tobe a dehydratase with broad substrate range having strong activity forthe dehydration of gluconate (Kim, S. Lee, S. B. Biotechnol. BioprocessEng. 2008, 13, 436).

TABLE 1 Enzymes used in this experiment and identity homology. Allexpressed in P. fluorescens pSGI WT/ Expression Organism (Vector) GeneID SYN Host Achromobacter 365 E3HJU7 Syn P. fluorescens (pRANGER)Achromobacter 359 #0385 wt P. fluorescens (pRANGER) Acinetobacter 360#0336 wt P. fluorescens (pRANGER) 359_Achromob 365_E3HJU7pSGI-360_Acinetobacter (SGI) 78 79 pSGI-359_Achromobacter (SGI) 95pSGI-365 Acromobacter

Proteins 359, 360, and 365 (SEQ ID NOs 33-35, respectively) showed 2-5mmole/min per mg of crude enzyme lysate activity for the synthesis ofdehydration of gluconate (gel not shown). pSGI-359 was isolated byprecipitation with ammonium sulfate and re-dissolving in buffer andassayed by the semicarbazide assay. Activities of 46.2 U/mL or 5.3 U/mg(1 unit=μmole/min) for the dehydration of gluconate were calculated fromsemicarbazide assay plots. Reaction buffer (93 mL) containing Kpi 10 mMpH 8.0 with 2 mM MgCl2 and 3.5 gr (0.016 mole) of sodium gluconate wasmixed with 7 mL of the previous gluconate dehydratase solution. Thereaction was incubated at 45° C. for 16 h before one aliquot wasanalyzed by HPLC-MS (FIG. 4). As shown in FIG. 4 one new major productwith the molecular weight of DHG was produced. The product was alsoshown to have activity with DHG dehydratases.

All proteins were cloned on the pRANGER™ (Lucigen, Middleton, Wis.)expression vector and were expressed in a Pseudomonas fluorecens strain.pRANGER™ is a broad host commercially available plasmid vectorcontaining the pBBR1 replicon, Kanamycin resistance and an pBAD promoterfor inducible expression of genes. For the enzyme assay a modificationof the semicarbazide assay for the quantification of alpha keto acid wasused to calculate the activity of each enzyme (Kim, S.; Lee, S. B.Biochem J. 2005, 387, 271). SEQ ID NOs: 30-32 and 33-35 show the aminoacid and nucleotide sequences, respectively, of the gluconatedehydratases #0385, #0336, and E3HJU7.

Example 2 Step 3—3-dehydro-gluconic acid (DHG) to (4S)-4,6-dihydroxy2,5-diketo hexanoate (2,5-DDH)

Enzymes of the family (EC 1.1.1.127) can be used to perform this step.Two examples are 2-dehydro-3-deoxy-D-gluconate 5-dehydrogenase and DHGdehydrogenases. Five enzymes from this family were cloned as shown inTable 2 below. pRANGER™ vector was used in every case.

TABLE 2 Cloned of DHG oxidoreductase (or 2-dehydro-3-deoxy-D-gluconate5-dehydrogenase) pSGI Gene Organism (Vector) ID WT/SYN Expression HostAgrobacterium 374 #9041 WT P. fluorescens sp (SGI) Agrobacterium 375#8939 WT P. fluorescens tumefaciens (SGI) E. coli 376 P37769 WT P.fluorescens Sphingomonas (SGI) 395 #5112 WT P. fluorescens Hoefleaphototrophica 396 #7103 WT P. fluorescens (SGI)

The product prepared from the dehydration of gluconate in Step 2 wasused as substrate for assaying the lysates of Table 2. As shown in thefollowing Table 3, enzymes were identified showing activity for theoxidation of DHG in assays measuring NADH formation (absorbance increaseat 340 nm).

TABLE 3 Activity calculations for oxidation of DHG to 2,5-DDH using DHGoxidoreductase. A unit = μmole/min of NADH U/mg (100 mM DHG) ENZ pH =7.5 pH = 8.5 (10 mM DHG) pH = 9.5 pSGI_395 0.012 0.070 (0.02)  0.120pSGI_396 0.033 0.139 (0.018) 0.418 pSGI_374 0.007 0.043 (0.012) 0.091pSGI_376 0.007 0.121 (0.01)  1.610

Further verification of the formation of 2,5-DDH by these enzymes wasshown in Step 16 where the reduction of 2,5-DDH (made from thedehydration of 5KGA) with pSGI-395 at acidic pH was shown.

Example 3 Steps 7 and 7B—Conversion of Guluronic Acid to D-Glucaric Acid(7) and Conversion of L-Iduronic Acid to Idaric Acid (7B)

To demonstrate Steps 7 and 7B the following study was performed. Uronatedehydrogenases (EC 1.1.1.203) are enzymes that oxidize glucuronic andgalacturonic acid. Three enzymes with sequence similarity to the knownuronate dehydrogenase (Expasy: Q7CRQ0; Prather, K. J, et al., J.Bacteriol. 2009, 191, 1565) were cloned from bacterial strains as shownin Tables 4 & 5.

TABLE 4 Cloned Uronate Dehydrogenases Organism pSGI (pET28) Gene IDExpression Agrobacterium #474 #8807 BL21DE3 Rhizobium #475 #8958 BL21DE3Pseudomonas #476 #1770 BL21DE3

TABLE 5 Sequence Identity #475 #476 Q7CRQ0 474_Agrobacterium 73 49 90475_Rhizobium 51 74 476_Pseudomonas 50

Each protein was expressed with a His tag from pET28 and was purifiedprior to their screening. Protein gels of the crude lysates and purifiedenzymes are shown in the gel of FIG. 1. After purification all enzymeswere tested for activity against glucuronate, as well as againstguluronate and iduronate. Kinetic measurements at different substrateconcentrations were performed and the calculated activities and Kmvalues for each enzyme are shown in Table 6. All enzymes showed goodactivity for glucuronate, and also for L-iduronate and guluronate.

TABLE 6 Activity and Km value for purified uronate dehydrogenases. Vmax(μM/min/mg); and Km (mM) Guluronate Enzyme Glucuronate Iduronate (Vmonly) 474 128.2; 0.37  0.96; 29.8 0.017 475 47.4; 0.22 0.59; 42.1 0.016476 90.9; 0.34 1.36; 29.6 0.014

Each plasmid shown in Table 4 was transformed in BL21DE3 E. coli cells.Clarified lysates were mixed with equal volume of (25 mL) ofequilibration buffer and purified on an Ni NTA column. Activity of eachpurified enzyme was measured in by mixing 0.050 mL of various dilutionsof each purified enzyme with 0.95 mL of reaction buffer (100 mM TrisHCl,pH 8.0, 50 mM NaCl, 0.75 mM NAD+). The reaction progress was measured bymonitoring of the formation of NADH at 340 nm. FIGS. 6 a and 6 b provideLineweaver-Burk plots for the oxidation of glucuronate and iduronate,with all three enzymes shown in FIG. 6. Clear positive slopes wereobtained with all enzymes giving the activities shown in the tableabove. Protein sequences of the uronate dehydrogenases are shown as SEQID NOs: 1-3 and the genes as SEQ ID NO: 4-6.

Pyrroloquinoline (PQQ) dependent aldehyde dehydrogenases also showedgood activity for the oxidation of both guluronate and iduronate. Theseare soluble periplasmic enzymes that were expressed in the E. colicytosol after their periplasmic target sequence was removed. Theactivities of crude lysates in units (μmole/min) per milligram of totallysate protein are shown in the following Table 6A. The actual activityof each enzyme is at least 2-5× higher if purified (see expression inFIG. 3).

Enzyme Iduronate U/mg Guluronate U/mg P75804 (SEQ ID NO: 73) 8.7 3.29522 (SEQ ID NO: 74) 7.3 6.1 6926 (SEQ ID NO: 75) 9.2 4.1 7510 (SEQ IDNO: 76) 7.3 3.7 7215 (SEQ ID NO: 77) 14.2 8.3 8386 (SEQ ID NO: 78) 4.31.5

The activities shown on Table 6A were measured using an artificialelectron acceptor DCPIP (2,6-dichloroindophenol) according to thefollowing protocol: In 0.95 mL of 20 mM Triethanol amine (pH 8.0)containing 0.2 mM DCPIP, 0.2 mM PMS (phnazine ethosulfate) and substrate(10-40 mM), 0.050 mL of enzyme (as crude lysate or 10-100× diluted withbuffer) is added and the reaction progress is followed by the change ofDCPIP absorbance at 600 nm. Because in their natural state these enzymesare transferring electrons to other proteins or cofactors in themembrane electron transport chain, the in vitro activity is measuredusing artificial electron acceptors with DCPIP being the most common.

The enzymes on Table 6A were active against a number of other aldehydesincluding butyraldehyde, butyraldehyde and glycerol (but not glucose).Therefore, these enzymes will oxidize the aldehyde group of iduronateand guluronate to give iduronic and glucaric acid respectively. In orderto confirm this selectivity, two of these enzymes, #403 and #412, wereexpressed in the periplasm of E. coli by fusing them with theperiplasmic target sequence of #403 (a native E. coli enzyme). Bothproteins were expressed in the periplasm but in lower levels compared tothe cytosol. The previous recombinant cells oxidized benzaldehyde tobenzoic acid in good yields and in lower yields produced glucaric andidaric acid from guluronate and iduronate.

Example 4 Step-15: Conversion of 5-Ketogluconate (5-KGA) to L-IduronicAcid (15) or Guluronic Acid (15A)

This example illustrates the identification of an enzyme capable ofisomerizing 5-KGA to iduronic acid (Step 15) or guluronic acid (Step15A). Thirteen enzymes from three different isomerase families werecloned as shown in Table 7, while their % sequence identity is shown inTable 8.

TABLE 7 Isomerases cloned Gene ID pSGI Archetype ® or EC Organism(pET28) Expasy WT/SYN 5.3.1.17 Rhizobium 433 #8938 WT 5.3.1.17 E. coli434 Q46938 (Expasy) WT 5.3.1.17 Rhizobium 435 #3891 WT 5.3.1.17Pannonibacter 436 #7102 WT 5.3.1.n1 Lactobacillus 458 A5YBJ4 (Expasy)SYN 5.3.1.n1 Acidophilum 440 F0J748 (Expasy) SYN 5.3.1.n1 Bacillus 437#9209 WT 5.3.1.n1 Ochrobactrum 438 #9732 WT 5.3.1.n1 Halomonas 439 #7403WT 5.3.1.12 Sphingobacteria 478 #1874 WT 5.3.1.12 Thermotoga 479 Q9WXR9SYN 5.3.1.12 Bacillus 480 Q9KFI6 SYN 5.3.1.12 Bacillus 481 O34808 SYN

TABLE 8 % Identities of isomerases EC 436 434 435 458 440 437 438 439481 480 479 478 433 5.3.1.17 65 44 43 16 13 18 11 14 6 11  11 7 4365.3.1.17 45 46 18 14 15 12 13 5 10  11 7 434 5.3.1.17 46 17 10 15 10 136 10  12 7 435 5.3.1.17 18 16 18 14 16 9 11  13 7 458 5.3.1.n1 37 57 4144 6 7  9 5 440 5.3.1.n1 40 67 50 6 6  6 5 437 5.3.1.n1 46 51 8 7 10 6438 5.3.1.n1 52 5 5  6 4 439 5.3.1.n1 6 7  8 5 481 5.3.1.12 7 36 54  4805.3.1.12  7 7 479 5.3.1.12 37  478 5.3.1.12

As shown in Table 8, enzymes with medium homology (underlined) withineach family were selected for cloning. The data demonstrated thatenzymes from all families showed activity for the isomerization of 5-KGAgiving L-iduronate as the main product. Two enzymes from the 5.3.1A7family (433 & 434) were also used in the example showing the formationof DDG from 5-ketogluconate (5KGA).

Activity for the isomerization of 5KGA and iduronate using enzymes fromTable 7 was measured using an enzymatic method that detected theformation of products by their activity against two different enzymes.For example, isomerization of 5KGA was detected by measuring theactivity of the product iduronate using uronate dehydrogenase(pSGI-476). Isomerization of iduronate was detected by measuring theactivity 5KGA reductase (pSGI-383, EC 1.1.1.69) of the product 5KGA.Presence of the products was also detected by GC-MS.

Enzymes from all families showed varying activity for the isomerizationof 5KGA and iduronate. Two enzymes from EC 5.3.1.12 were used in a cellfree reaction to isomerize 5KGA and ultimately produce DDG as describedin the example. The enzymes were purified and showed a single band bygel electrophoresis. The purified isomerases were used in reactionsusing lysate and buffer containing 5KGA or Iduronate. Product formationwas demonstrating using both HPLC and the previously described enzymaticmethods. Results for 17 h of incubation using both HPLC and enzymeassays are shown in FIG. 7 a. All enzymes showed good activity for theisomerization of both 5KGA and iduronate. Yields for iduronateisomerization by pSGI433, pSGI 434, pSGI 435, and p SGI 436 were 56%,48% 42%, (436 not measured), respectively when measured enzymaticallyand 78.8%, 78.5%, 73.3% and 76.6%, respectively when measured by HPLCassay. Yields after 16 h for 5KGA isomerization by the same enzymes were18%, 17%, and 19% respectively (436 not measured) when measured byenzymatic assay, and 16.6%, 17.8%, 16.3%, and 16.9%, respectively, whenmeasured by HPLC assay.

EC 5.3.1.12 Enzymes

Enzymes from the EC 5.3.1.12 family (glucuronate isomerases) were alsopurified by gel electrophoresis, isolated, and used to prepare reactionsby mixing with buffer (50 mM HEPES, 1 mM ZnC12, pH 8.0) that contained 5mM of 5KGA or Iduronate. The reactions were incubated at 30° C. andanalyzed for product formation using both HPLC and enzymatic methods.Results are shown in FIG. 7 b.

5.3.1.17 Enzymes

Enzymes pSGI-478 and pSGI-479 (5-dehydro-4-deoxy-D-glucuronateisomerases) showed isomerization activity for both 5KGA and iduronate.This activity was also confirmed with the enzymatic assays as above.Yields for isomerization of iduronate by pSGI-478 and -479 were 50% and37%, respectively, when measured enzymatically, and 20% and 18% whenmeasured by HPLC. Yields for 5KGA isomerization were 23% and 26%,respectively, when measured enzymatically, and 24% and 16%, respectivelywhen measured by HPLC. Results are shown in FIG. 7 a.

5.3.1.n1 Enzymes

Enzymes in this family were purified by gel electrophoresis. Productformation was measured using enzymatic assays as described above and theresults are shown in FIG. 8. All enzymes cloned in this family wereshown to have activity for the isomerization of 5KGA and iduronate.

In each case plasmids were transformed in BL21DE3 and proteins purifiedon a Ni NTA column.

Example 5 Step 16—5-Keto-Gluconate (5KGA) to (4S)-4,6-Dihydroxy2,5-Diketo Hexanoate (2,5-DDH)

The three gluconate dehydratases described in Step 2 (Example 1) wereexpressed as described in Example 1, along with a purified glucaratedehydratase from Step 8. Enzymatic reactions for activity were performedand HPLC-MS analysis showed the formation of 2,5-DDH (FIG. 9), which wasalso confirmed by the fact that formation of the new product wasaccompanied by the reduction of 5-KGA only in the samples containinggluconate dehydratases, as well as by enzymatic assays with DHGdehydratase (pSGI-395). Good slopes at 340 nm indicating large enzymeactivity were obtained when NADH, pSGI-395 lysate and aliquots of theprevious reactions were mixed (data not shown). This result incombination with the HPLC analysis prove that the gluconate dehydratasesexamined dehydrate 5KGA to 2,5-DDH.

Example 6 Step 19—Conversion of 1,5-Gluconolactone to Guluronic Acidδ-Lactone

1,5-gluconolactone oxidation is a side activity of enzymes from thealditol oxidases (EC 1.1.3.41) family. These enzymes oxidize variousalditols such as sorbitol, xylitol, glycerol and others. Enzymes wereidentified having activity for the oxidation of 1,5-gluconolacone, asshown in Table 6 below.

TABLE 6 Alditol oxidases with activity on 1,5-gluconolactone.1,5-Gluconolactone Reaction Setup Sorbitol Enzyme Substrate EnzymeEnzyme Source U/mg U/mg mg mg/mM Yield AO#13 Terriglobuds roseus 0.230.02 5.3 15/85 7% AO#22 Granulicella mallensis 0.27 0.015 7.6 15/85 9%AO#28 Streptomyces acidiscabies 1.30 0.010 15 15/85 8% AO#36Actinomycetales (SGI) 1.83 0.102 25 90/35 46%  AO#51 Frankia sp 0.590.019 NT NT NT AO#57 Propionibacteriacaeae (SGI) 1.47 0.051 40 70/57 6%AO#76 Streptomyces sp. 1.45 0.045 8.2 15/85 23%  AO#251* Paenibacillussp. 0.47 0.003 24  15 8.5 ~2%   *crude lysate

Reactions were prepared using lysates of all the purified enzymes shownon Table 6. Reactions were prepared in 50 mM K-phosphate buffer, pH 7.0with 0.5 mg/mL catalase and incubated at 30° C. A new product wasobserved by HPLC-MS analysis showing the same retention time asguluronate after comparison with authentic standards (FIG. 10). This wasconfirmed by GC-MS, where the product also had the same MS fingerprintas guluronate. It is therefore clear that all the alditol oxidasesdescribed in the Table oxidize the 6-OH of 1,5-gluconolactone to producethe guluronic acid lactone. All alditol oxidases were cloned in pET28awith a HisTag and were expressed in BL21DE3 and purified on a Ni NTAcolumn.

Example 7 Synthesis of FDCA and Other Intermediates

Purified DDG mono potassium salt was used for the dehydration to2,5-FDCA. Sulfuric acid was added to DDG and the reaction stirred at 60°C. The in situ yield was calculated (by HPLC-MS) to be ˜24% and ˜27%.

The reaction solutions were combined and then diluted by pouring intoice (to neutralize the heat). Approximately equivalent volume of THF wasadded, and the solution transferred to a separation funnel. Sodiumchloride salt was added until separation was achieved. The solution wasagitated between additions for best possible dissolution. The aqueouslayer was removed, and the THF layer washed 3× more with sat. NaCLsolution. Sodium sulfate was added and the solution left sittingovernight. Two layers formed again overnight. The aqueous layer wasdiscarded and then silica gel was added to the solution. It was thenconcentrated down to solids via rotovap. The solids were loaded into asilica flash column and then separated via chromatographically. Thefraction was concentrated and dried. The isolated yield was 173.9 mg.Corrected yield: 24.9%. ¹H and ¹³C NMR and HPLC-MS analysis confirmedthe product

Dehydration of DDG Dibutyl-2,5-FDCA in BuOH/H₂SO₄

Dehydration of un-derivitized lyophilized DDG containing the dehydrationsalts in BuOH was done using a Dean-Stark apparatus. Under theseconditions, DDG was added to BuOH, and then H2SO4 was added and thereaction heated at 140° C. After stirring for 4 h HPLC-MS analysis showsthe disappearance of DDG and the formation of dibutyl-2,5-FDCA. The insitu yield was calculated (by HPLC-MS) to be 36.5%.

The mixture was extracted with water, 1% NaOH, and again with water.Then the organic layer was concentrated to a final mass of 37.21 g. Aportion of this mass (3.4423 g) was removed and 0.34 g ofdibutyl-2,5-FDCA was purified using HPLC. Extrapolating the yield of theisolated product to the total amount of compound isolated from thereaction (37.21 g) and taking into account the amount of salts presentin the original DDG (˜60% pure by weight) the reaction yield wascalculated to be 42%. ¹H and ¹³C NMR and HPLC-MS analysis confirmed theproduct

Synthesis of Dibutyl DDG

In another aspect the invention provides a method for synthesizing aderivative of DDG. The method involves contacting DDG with an alcohol,an inorganic acid, and optionally a co-solvent to produce a derivativeof DDG. Optionally the derivative of DDG can be purified. The reactioncan have a yield of the derivative of DDG of at least 10% molar yield orat least 15% molar yield or at least 20% molar yield or at least 25% orat least 30% or at least 35% molar yield or at least 40% molar yield.The inorganic acid can be sulfuric acid and the alcohol can be methanol,ethanol, propanol, butanol, isobutanol, or any C1-C20 alcohol. Invarious embodiments the co-solvent can be any of THF, acetone,acetonitrile, an ether, butyl acetate, an dioxane, chloroform, methylenechloride, 1,2-dichloroethane, a hexane, toluene, and a xylene. When thealcohol is ethanol the DDG derivative will be DDG mono-ethyl esterand/or DDG diethyl ester. When the alcohol is butanol the DDG derivativewill be DDG mono-butyl ester and/or DDG dibutyl ester.

DDG mono-potassium salt was used for derivatization according to thefollowing protocol. In a 1 L Morton type indented reaction vesselequipped with a mechanical stirrer and heating mantle was charged with60:40 DDG:KCl (31.2 mmol), BuOH, and heptane. In a separate vial,sulfuric acid was added to water, and allowed to cool after dissolution.The solution was then added to the flask. The solution was kept at 30°C.

The precipitate was filtered off concentrated. The remaining gel wasdissolved in EtOAc, and then TLC plates were spotted with the solutionsand the plates were sprayed with a phosphomolybdic acid mixture, andthen heated to at least 150° C. on a hot plate to identify the DDG-DBEfraction. Isolated yield: 4.62 g (15.2 mmol, 47% yield), >98% purity.¹E1 and ¹³C NMR and HPLC-MS analysis confirmed the product.

Different solvents can be used in the synthesis of DDG esters, such asmixtures of BuOH (5%-95% v/v) with co-solvents such as THF, acetone,acetonitrile, ethers (dibutyl, diethyl etc), esters such asButyl-acetate, 1,6-dioxane, chloroform, methylene chloride,1,2-dichloroethane, hexanes, toluene, and xylenes may be used ascosolvents. Reaction catalysts such as acids (sulfuric, hydrochloric,polyphosphoric or immobilized acids such as DOWEX) or bases (pyridine,ethyl-amine, diethyl-amine, boron trifluoride) or other catalystscommonly used for the esterification of carboxylic acids.

Dehydration of Dibutyl-DDG to Dibutyl-FDCA in n-BuOH/H₂SO₄

A stock solution of DDG-DBE (di-butyl ester) was made in butanol andtransferred to a clean, dry 100 mL round-bottomed flask equipped with astir bar. To the flask, 25 mL of conc. sulfuric acid was added. Theflask was sealed and then stirred at 60° C. for 2 hrs. The in situ yieldwas calculated to be ˜56%. The reaction solution was concentrated andthe residue was dissolved in MTBE and transferred to a separationfunnel, and then washed with water. The recovered organic layer wasconcentrated and then separated via HPLC for an isolated yield: 250.7 mg(˜90% purity) and 35% isolated yield (corrected for purity). ¹C and ¹³CNMR and HPLC-MS analysis confirmed the product.

Example 8 Cell Free Synthesis of DDG and FDCA and Derivatives from 5-KGA(Route 2A)

This example illustrates the enzymatic conversion of 5KGA to DDG usingpurified enzymes according to Scheme 6 (a sub-Scheme of 2B), and alsoillustrates the DDG produced being dehydrated to FDCA using chemicalsteps. The Scheme involves the steps of isomerization of 5KGA (Step 15)and the subsequent oxidation to idaric acid (Step 7B). DDG was alsodehydrated under differing chemical conditions to FDCA. The last step(Step-8A) was performed using glucarate dehydratase from E. coli.

Scheme 6 is illustrated in FIG. 11. The scheme was performed using acell free enzymatic synthesis of DDG from 5-KGA. The Scheme involves theperformance of steps 15, 7B and 8A (see FIG. 2 d). Two additionalproteins were used to complete the reaction path, the first beingNADH-oxidase (Step A) that is recycling the NAD+ cofactor in thepresence of oxygen, and catalase (Step B) that decomposes the peroxideproduced from the action of NADH oxidase. The enzymes are shown in thefollowing Table 7. All enzymes contained a HisTag and were purifiedusing an Ni-NTA column. Yields for this synthesis of DDG were calculatedto be at least 88-97%.

TABLE 7 STEP Enzyme EC Organism 15 pSGI-433 5.3.1.17 Rhizobium (SGI)(DTHU_IS) 15 pSGI-434 5.3.1.17 E. coli (DTHU_IS)  7B pSGI-476 1.1.1.203Pseudomonas (SGI) (UroDH)  8A pSGI-353 4.2.1.40 E. coli (GlucDH) ApSGI-431 1.6.3.1 Thermus (NADH_OX) thermophiilus B Catalase 1.11.1.6Corynbacterium

500 mL of liquid culture was purified for each isomerase for thereaction. Besides the enzymes shown on Table 7, each reaction contained50 mM TrisHCl (pH 8.0), 50 mM NaCl, 1 mM ZnCl₂ and 2 mM MgCl₂, 1 mMMnCl₂ and 1 mM NAD⁺. Reactions were analyzed by HPLC after 16 h ofincubation and FIG. 12 presents the chromatograms.

For dehydration to FDCA, the reaction mixtures of both samples werecombined and lyophilized into a white powder, which was split into twosamples and each dissolved in AcOH with 0.25M H₂SO₄ or in 4.5 mL BuOHwith 0.25M H₂SO₄. Both reactions were heated in sealed vials for 2-4 hat 120° C. Reaction products are shown in FIG. 13.

Samples 1 and 2 represent authentic standard and the 3 h time point fromthe reaction in AcOH/H₂SO₄, respectively. Spiking of sample 2 withsample 1 gave a single peak further verifying the FDCA product. Samples1 and 3 (FIG. 13) represent authentic standard and the 4 h time pointfrom the reaction in BuOH/H₂SO₄, respectively. The formation of FDCAfrom the enzymatic reactions further confirms the presence of DDG inthese samples.

Example 9 Synthesis of DDG from Glucose and Gluconate

This example shows the enzymatic conversion of glucose and gluconate toDDG. The reaction was conducted with purified enzymes, and crude lysatesas a catalyst. Enzymes and substrates were combined in a bio-reactor asshown in the Table below:

ST-14 ST-15 ST-7B ST-8A ST-A Substrate ST-1 pSGI-504 pSGI-434 pSGI-476pSGI-353 pSGI-431 ST-B Rxn-1 Glucose 2 mg 7 mL¹ 50 mL² 7.5 mL¹ 1 mL³ 4mL⁴ 2 mg 600 mg Rxn-2 Gluconate — 7 mL 50 mL 7.5 mL 1 mL 4 mL 2 mg 700mg ¹Lysate from 500 mL liquid culture of recombinant E. coli withplasmid ²Lysate from 2 L liquid culture of BL21DE3/pSGI-434 ³Purifiedenzyme, ~30 Units of activity (or 3 mg of purified GlucD) ⁴Lysate from250 mL of culture

The reaction was incubated at 35° C. and dissolved oxygen and pH werekept at 20% and 8 respectively. Time points were analyzed by HPLC-MS andthe results are shown in FIG. 17 b. Extracted chromatograms verified theDDG mass (not shown) and corresponding MS fragmentation. The resultsclearly showed production of DDG during incubation of the enzymes witheither glucose or gluconate.

Example 10 Construction of Expression Cassettes for RecombinantGlucarate Dehydratases

The following example describes the creation of recombinant nucleic acidconstructs that contained coding sequence of a D-glucarate dehydrataseactivity (GDH, EC 4.2.1.40) for heterologous expression in E. colicells.

Genes encoding D-Glucarate dehydratase from E. coli (Expasy: P0AES2),Acinetobacter ADP1 (Expasy: P0AES2), as well as a proprietaryPseudomonas bacterial strain (#8114) were PCR-amplified from genomicDNA.

Each of the PCR-amplified genes was subsequently cloned into thebacterial transformation vector pET24a(+), in which the expression ofeach of the GDH genes was placed under control of a T7 promoter. Thenucleotide sequences of each of the PCR-amplified inserts were alsoverified by sequencing confirmation.

Example 11 E. coli Strains Expressing Recombinant Glucarate Dehydratases

Each of the expression vectors constructed as described in Example 9 wasintroduced into NovaBlue(DE3) E. coli by heat shock-mediatedtransformation. Putative transformants were selected on LB agarsupplemented with Kanamycin (50 μg/ml). Appropriate PCR primers wereused in colony-PCR assays to confirm positive clones that contained eachof the expression vectors.

For each expression vector, a bacterial colony was picked fromtransformation plates and allowed to grow at 30° C. in liquid LB mediasupplemented with Kanamycin (50 μg/ml) for two days. The culture wasthen transferred into vials containing 15% glycerol and stored at −80°C. as a frozen pure culture.

Example 12 Demonstration of In Vitro Synthesis of DDG by Using CellLysate of Recombinant E. coli Cells Expressing a GDH Enzyme

This Example describes how in vitro synthesis of DDG intermediate wasachieved using recombinant glucarate dehydratase (GDH) enzymes producedin E. coli cells.

Preparation of Cell Lysates:

Recombinant bacterial strains constructed as described previously inExample 2 were grown individually in 3 mL of liquid LB mediasupplemented with Kanamycin (50 μg/ml) at 30° C. on a rotating shakerwith rotation speed pre-set at 250 rpm for 1 day. This preculture wasused to inoculate 100 mL of TB media containing Kanamycin (50 ug/ml),followed by incubation at 30° C. on a rotating shaker pre-set at 250 rpmfor 2-3 hour until early log phase (OD₆₀₀˜0.5-0.6) before isopropyl D-1thiogalactopyranoside (IPTG; 0.25 mM final concentration) was added toinduce protein expression. Cells were allowed to grow for another 18hours at 30° C. before they were harvested by centrifugation,resuspended in 15 mL of lysis buffer (10 mM phosphate buffer, pH 7.8, 2mM MgCl₂) and were lysed by sonication. The production of recombinantenzymes in E. coli cells was quantified using standard pre-cast SDS-PAGEgels system (BioRad), and specific activity was measured according to aprocedure described by Gulick et al. (Biochemistry 39, 4590-4602, 2000).Crude cell lysates or purified enzymes (using the HisTag) were thentested for the ability to convert gram amounts of glucarate to DDG asdescribed in greater detail below.

Enzymatic Dehydration of Glucarate

A large scale oxidation of glucarate using glucarate dehydratase wasprepared. 350 mL of water 25 g of glucaric acid sodium salt (0.1 mole)and 4.5 gr of KOH (0.8 mole) were mixed in an Erlenmyer flask. Residualsolid glucarate was dissolved by the slow addition of 5M KOH solution(˜3 mL) and the pH was adjusted to 7.4. In this solution 100 mg ofpurified glucarate dehydratase and 2 mM MgCl2 were added, and themixture was placed in an orbital shaker at 30° C. for 20 h. The next daythe precipitate is removed by filtration. The pH of the reaction wasessentially unchanged. Analysis of the reaction revealed the presence ofonly DDG in the solution, indicating >95% yield.

Purification of DDG Product from Enzymatic Reactions:

DDG produced via enzymatic dehydration was purified by using either ofthe two following techniques. The enzymatic dehydration reactions wereacidified to pH˜3.0 with 6M HCl, filtered to eliminate precipitate, andsubsequently lyophilized to produce a white powder consisting of DDG andsalts. The same DDG purity (but lower amount of salts) can be obtainedif the reaction was filtered through a 10 KDa membrane to removeproteins and then lyophilized. Without any further purification bothprevious lyophilized powders can be dehydrated to FDCA (or its esters)or can be esterified to dibutyl DDG as shown in other examples of thisapplication.

Results of HPLC-MS analyses indicated that DDG product constituted atleast 95% of the total products in the samples obtained from either ofthe two purification techniques.

Example 13 Demonstration of In Vitro Synthesis of FDCA from DDG inOne-Step Chemical Reaction

Applicants have discovered that the synthesis of FDCA (i.e. the freeacid form) could be achieved by a chemical conversion of DDG to FDCA inthe presence of H₂SO₄. The reaction was performed as follows.Approximately 20 mg of DDG acid (crude lyophilized powder with saltspreviously purified as described in Example 3) and 0.25 M of H2SO4 wereadded into an air tight sealed tube containing 1 mL of water and 1 mL ofDMSO. The DDG was found completely dissolved in this solution. Thereaction was stirred at 105° C. for 18 hours. Results of an HPLC-MSanalysis performed on a crude reaction sample indicated the formation ofFDCA free acid (FDCA: 2,5-furan dicarboxylic acid) as the major product,as well as insignificant amounts of some other unidentified byproducts.As a control in HPLC-MS analysis, a commercial FDCA was analyzed in thesame conditions.

Example 14 Demonstration of In Vitro Synthesis of FDCA-Esters(Dimethyl-, Diethyl-, Dibutyl-, and Isopropyl-Esters)

Synthesis of Diethyl-2,5 FDCA from Purified DDG:

In an air tight sealed tube, 18 mL of EtOH, 0.2 gram (1 mmole) of DDGacid, previously purified as described in Example 11, and 0.25 M ofH₂SO₄ were added. The DDG acid was not completely dissolved in thissolution. The reaction was gently stirred at 105° C. for 18 hours.Results of a GC-MS analysis of a crude reaction sample indicated thatthe formation of diethyl-FDCA the major product. As a control, anauthentic FDCA was chemically synthesized, esterified to diethyl-FDCAand analyzed in the same conditions.

Example 15 Synthesis of Dibutyl-2,5 FDCA from Purified DDG

In an air tight sealed tube, 18 mL of n-BuOH, 0.2 gram (1 mmole) of DDGacid, previously purified as described in Example 11, and 0.25 M ofH₂SO₄ were added. The DDG acid was not completely dissolved in thissolution. The reaction was gently stirred at 105° C. for 18 hours. Asshown in FIG. 15, results of the GC-MS analysis of a reaction sampleindicated that diethyl-FDCA (FDCA: 2,5-furan dicarboxylic acid) wasformed as the major product. As a control, an authentic FDCA waschemically synthesized, esterified to diethyl-FDCA, and analyzed in thesame conditions.

Example 16 Synthesis of Dibutyl-2,5 FDCA from Crude DDG (Unpurified)

0.2 gram (1 mmole) of crude DDG acid, which was an unpurifiedlyophilized powder obtained directly from the enzymatic dehydration ofglucarate as described in Example 11, was added into an air tight sealedtube containing 18 mL of n-BuOH, followed by addition of 0.25 M ofH₂SO₄. The crude DDG acid was not completely dissolved in this solution.The reaction was gently stirred at 105° C. for 18 hours. Results of aGC-MS analysis of a crude reaction sample indicated that diethyl-FDCA(FDCA: 2,5-furan dicarboxylic acid) was formed as the major product. TheGC-MS result indicated that the present of contaminant salts incrude/unpurified lyophilized powder did not significantly affect thereaction outcome. As a control, an authentic FDCA was chemicallysynthesized, esterified to diethyl-FDCA, and analyzed in the sameconditions.

Example 17 In Vitro Production of FDCA and/or Esters Using ImmobilizedAcids

In industrial practices, immobilized acids offer many advantages forperforming dehydrations since they can typically operate in severaltypes of solvent (aqueous, organic or mixed, etc.). In addition, theycan be easily recycled and be re-used. Following some examples of thesynthesis of esters of FDCA using immobilized AMBERLYST®15 (Rohm andHaas, Philadelphia, Pa.) and DOWEX®50 WX8 (Dow Chemical Co, Midland,Mich.).

Synthesis of Dibutyl-FDCA from Crude DDG by Using DOWEX®50 WX8

In an air tight sealed tube, 2 mL of n-Butanol, 20 mg of crude DDG acid(unpurified lyophilized powder containing salts) and 200 mg of DOWEX®50WX8 were combined. The DDG was not completely dissolved in thissolution. The reaction was gently stirred at 105° C. for 18 hours.Results of the GC-MS analysis of a crude reaction sample indicated thatdiethyl-FDCA (FDCA: 2,5-furan dicarboxylic acid) was formed as the majorproduct. This GC-MS result indicated that the present of contaminantsalts (phosphate and NaCl) in crude/unpurified lyophilized powder didnot significantly affect the reaction outcome. As a control, anauthentic FDCA was chemically synthesized esterified to diethyl-FDCA andanalyzed in the same conditions.

Synthesis of Dibutyl-FDCA from Crude DDG by Using AMBERLYST®15

In an air tight sealed tube, 2 mL of n-Butanol, 20 mg of crude DDG acid(crude lyophilized powder with salts) and 200 mg of AMBERLYST®15 (Rohmand Haas, Philadelphia, Pa.) were combined. The DDG was not completelydissolved in this solution. The reaction was gently stirred at 105° C.for 18 hours. Results of the GC-MS analysis of a crude reaction sampleindicated that diethyl-FDCA (FDCA: 2,5-furan dicarboxylic acid) wasformed as the major product. This GC-MS result indicated that thepresent of contaminant salts (phosphate and NaCl) in crude/unpurifiedlyophilized powder did not significantly affect the reaction outcome. Asa control, an authentic FDCA was chemically synthesized esterified todiethyl-FDCA and analyzed in the same conditions.

Synthesis of Ethyl-FDCA from Crude DDG by Using AMBERLYST®15

In an air tight sealed tube, 2 mL of ethanol, 20 mg of crude DDG acid(unpurified lyophilized powder containing salts) and 200 mg ofAMBERLYST®15 (Rohm and Haas, Philadelphia, Pa.) were combined. The DDGwas not completely dissolved in this solution. The reaction was gentlystirred at 105° C. for 18 hours. Results of the GC-MS analysis of acrude reaction sample indicated that diethyl-FDCA (FDCA: 2,5-furandicarboxylic acid) was formed as the major product. This GC-MS resultindicated that the present of contaminant salts (phosphate and NaCl) incrude/unpurified lyophilized powder did not significantly affect thereaction outcome. As a control, a commercial FDCA was chemicallyesterified to diethyl-FDCA and analyzed in the same conditions.

Synthesis of Diethyl-FDCA from Crude DDG by Using DOWEX®50 WX8

In an air tight sealed tube, 2 mL of ethanol, 20 mg of crude DDG acid(unpurified lyophilized powder containing salts) and 200 mg of DOWEX®50WX8 were combined. The DDG was not completely dissolved in thissolution. The reaction was gently stirred at 105° C. for 18 hours.Results of the GC-MS analysis of a crude reaction sample indicated thatdiethyl-FDCA (FDCA: 2,5-furan dicarboxylic acid) was formed as the majorproduct. This GC-MS result indicated that the present of contaminantsalts (phosphate and NaCl) in crude/unpurified lyophilized powder didnot significantly affect the reaction outcome. As a control, acommercial FDCA was chemically esterified to diethyl-FDCA and analyzedin the same conditions.

Example 18 Production of FDCA Derivatives

The synthesis of a number of high-value FDCA derivatives is described inFIG. 16 in which dehydration of DTHU produces furfural-5-carboxylicacid, i.e. FCA, which is then chemically or enzymatically oxidized toFDCA, be reduced to FCH, or be transaminated (using chemical reductiveamination or transaminase) to amino acid-AFC.

Example 19 Production of Di-Butyl FDCA in a Gas Phase Reaction

In this example the inlet of the GC was used as a high temperaturereactor to catalyze the dehydration of di-butyl DDG to di-butyl FDCA.The resulting products were chromatographically separated detected bymass spectrometry. A solution of di-butyl DDG (10 mM) and sulfuric acid(100 mM) in butanol was placed in a GC vial. The vial was injected intoa GC and FDCA Dibutyl ester was observed. The reaction occurred in the300° C. inlet (residence time=4 seconds). The average yield of 6injections was 54%.

GC Settings: Direct Liquid Inject/MS Detector

Inlet: 300° C., total flow 29.51 ml/min, split ratio 10:1, split flow24.1 ml/min, Septum Purge flow 3 mL/min.

GC liner: 4 mm, glass wool (P/N 5183-4647)

Column Flow: 2.41 ml/min He constant pressure control

Oven Program: At 40° C. hold for 2 min, then ramp 25° C./min to 275° C.,then ramp 40° C./min to 325° C., hold for 2 min.

Column: HP-5MS, Agilent Technologies, 30 m×0.25 mm×0.25 um.

Total Runtime: 14.65 minutes

MSD Transfer line: 290° C.

MS Source: 250° C.

MS Quad: 150° C.

Retention Times:

2,3-FDCA Dibutyl ester: 9.3 min

2,5-FDCA Dibutyl ester: 9.7 min

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

No admission is made that any reference constitutes prior art. Thediscussion of the references states what their authors assert, and theapplicants reserve the right to challenge the accuracy and pertinence ofthe cited documents. It will be clearly understood that although anumber of prior art publications are referred to herein, this referencedoes not constitute an admission that any of these documents fowls partof the common general knowledge in the art.

It should also be understood that the foregoing examples are offered toillustrate, but not limit, the invention.

What is claimed is:
 1. A method for producing a product of an enzymaticor chemical pathway from a starting substrate, the pathway comprisingone or more conversion steps selected from the group consisting of: anenzymatic conversion of guluronic acid into D-glucarate (Step 7); anenzymatic conversion of 5-ketogluconate (5-KGA) into L-Iduronic acid(Step 15); an enzymatic conversion of L-Iduronic acid into Idaric acidStep 7b); and an enzymatic conversion of 5-ketocluconate into4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16); an enzymaticconversion of 1,5-gluconolactone to gulurono-lactone (Step 19).
 2. Themethod of claim 1 wherein the one or more conversion steps is theenzymatic conversion of guluronic acid into D-glucarate (Step 7).
 3. Themethod of claim 1 wherein the one or more conversion steps is theenzymatic conversion of 5-ketogluconate (5-KGA) into L-Iduronic acid(Step 15).
 4. The method of claim 1 wherein the one or more conversionsteps is the enzymatic conversion of L-Iduronic acid into Idaric acidStep 7b).
 5. The method of claim 1 wherein the one or more conversionsteps is the enzymatic conversion of 5-ketogluconate into 4,6-dihydroxy2,5-diketo hexanoate (2,5-DDH) (Step 16).
 6. The method of claim 1wherein the one or more conversion steps is the enzymatic conversion of1,5-gluconolactone to gulurono-lactone (Step 19).
 7. The method of claim1 wherein the product of the enzymatic pathway is5-dehydro-4-deoxy-glucarate (DDG).
 8. The method of claim 1 wherein thesubstrate is glucose and the product is 5-dehydro-4-deoxy-glucarate(DDG), comprising the steps of: the enzymatic conversion of D-glucose to1,5-gluconolactone (Step 1); the enzymatic conversion of1,5-gluconolactone to gulurono-lactone (Step 19); the enzymaticconversion of gulurono-lactone to guluronic acid (Step 18); theenzymatic conversion of guluronic acid to D-glucarate (Step 7); theenzymatic conversion of D-glucarate to 5-dehydro-4-deoxy-glucarate (DDG)(Step 8).
 9. The method of claim 1 wherein the substrate is glucose andthe product is DDG, comprising the steps of: the conversion of D-glucoseto 1,5-gluconolactone (Step 1); the conversion of 1,5-gluconolactone togluconic acid (Step 1a); the conversion of gluconic acid to5-ketogluconate (5-KGA) (Step 14); the conversion of 5-ketogluconate(5-KGA) to L-Iduronic acid (Step 15); the conversion of L-Iduronic acidto Idaric acid (Step 7b); and the conversion of Idaric acid to DDG (Step8a).
 10. The method of claim 1 wherein the substrate is glucose and theproduct is DDG, comprising the steps of: the conversion of D-glucose to1,5-gluconolactone (Step 1); the conversion of 1,5-gluconolactone togluconic acid (Step 1a); the conversion of gluconic acid to5-ketogluconate (5-KGA) (Step 14); the conversion of 5-ketogluconate(5-KGA) to 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16); theconversion of 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) to4-deoxy-5-threo-hexosulose uronate (DTHU) (Step 4); and the conversionof 4-deoxy-5-threo-hexosulose uronate (DTHU) to DDG (Step 5).
 11. Themethod of claim 1 wherein the substrate is glucose and the product isDDG, comprising the steps of: the conversion of D-glucose to1,5-gluconolactone (Step 1); the conversion of 1,5-gluconolactone togluconic acid (Step 1a); the conversion of gluconic acid to5-ketogluconate (5-KGA) (Step 14); the conversion of 5-ketogluconate(5-KGA) to L-Iduronic acid (Step 15); the conversion of L-Iduronic acidto 4-deoxy-5-threo-hexosulose uronate (DTHU) (Step 7b); and theconversion of 4-deoxy-5-threo-hexosulose uronate (DTHU) to DDG (Step 5).12. The method of claim 8 further comprising the step of converting theDDG to 2,5-furan-dicarboxylic acid (FDCA).
 13. The method of claim 9further comprising the step of converting the DDG to2,5-furan-dicarboxylic acid (FDCA).
 14. The method of claim 10 furthercomprising the step of converting the DDG to 2,5-furan-dicarboxylic acid(FDCA).
 15. The method of claim 11 further comprising the step ofconverting the DDG to 2,5-furan-dicarboxylic acid (FDCA).
 16. The methodof claim 12 wherein converting the DDG to FDCA comprises contacting DDGwith acid to convert the DDG to FDCA.
 17. The method of claim 13 whereinconverting the DDG to FDCA comprises contacting DDG with acid to convertthe DDG to FDCA.
 18. The method of claim 14 wherein converting the DDGto FDCA comprises contacting DDG with acid to convert the DDG to FDCA.19. The method of claim 15 wherein converting the DDG to FDCA comprisescontacting DDG with acid to convert the DDG to FDCA.
 20. A method forsynthesizing a derivative of FDCA comprising: contacting DDG with analcohol, an inorganic acid at a temperature in excess of 60 C to formFDCA.
 21. The method of claim 20 wherein the alcohol is butanol orethanol.
 22. The method of claim 20 having a yield of at least 25%molar.
 23. A method of synthesizing a derivative of DDG comprising:contacting DDG with an alcohol, an inorganic acid, and optionally aco-solvent to produce a derivative of DDG.
 24. The method of claim 23wherein: a) the alcohol is ethanol or butanol; b) the inorganic acid issulfuric acid; and c) the co-solvent is selected from the groupconsisting of: THF, acetone, acetonitrile, an ether, ethyl acetate,butyl acetate, an dioxane, chloroform, methylene chloride,1,2-dichloroethane, a hexane, a heptane, toluene, carbon tetrachloride,petroleum ether, and a xylene.
 25. A method for synthesizing derivativeof FDCA comprising: contacting a derivative of DDG with an inorganicacid to produce a derivative of FDCA.
 26. The method of claim 25 havinga yield of greater than 25% molar.
 27. The method of claim 26 whereinthe derivative is DDG is selected from the group consisting: methyl-DDG,ethyl-DDG, butyl-DDG, di-methyl DDG, di-ethyl-DDG, and di-butyl DDG. 28.The method of claim 25 further comprising that the derivative of FDCA isde-esterified to yield FDCA.
 29. The method of claim 25 furthercomprising a step of polymerizing the derivative of FDCA.
 30. A methodfor synthesizing FDCA comprising: contacting DDG with an inorganic acidin a gas phase.
 31. A method for synthesizing FDCA comprising:contacting DDG with an inorganic acid at a temperature in excess of 120C.
 32. A method for synthesizing FDCA comprising: contacting DDG with aninorganic acid under anhydrous reaction conditions.
 33. A method forproducing a product of an enzymatic or chemical pathway from a startingsubstrate, the pathway comprising one or more conversion steps selectedfrom the group consisting of: the conversion of DTHU to DDG (Step-5);the conversion of gluconic acid to guluronic acid (Step-6); theconversion of DEHU to DDH (Step 7A). the conversion of guluronic acid toDEHU (Step 17A);
 34. The method of claim 33 wherein the substrate isglucose and the product is DDG, comprising the steps of: the conversionof D-glucose to 1,5-gluconolactone (Step 1); the conversion of1,5-gluconolactone to gluconic acid (Step 1a); the conversion ofgluconic acid to 3-dehydro-gluconic (DHG) (Step-2) the conversion of3-dehydro-gluconic (DHG) to 4,6-Dihydroxy 2,5-diketo hexanoate (2,5-DDH)(Step-3) the conversion of 2,5 DDH to 4-deoxy-5-threo-hexosulose uronate(DTHU) (Step 4) the conversion of DTHU to DDG (Step-5).
 35. The methodof claim 33 wherein the substrate is glucose and the product is DDG,comprising the steps of: the conversion of D-glucose to1,5-gluconolactone (Step 1); the conversion of 1,5-gluconolactone togluconic acid (Step 1a); the conversion of gluconic acid to guluronicacid (Step-6) the conversion of guluronic to glucarate (Step-7) theconversion of glucarate to DDG (Step-8)
 36. The method of claim 33wherein the substrate is glucose and the product is DDG, comprising thesteps of: the conversion of D-glucose to 1,5-gluconolactone (Step 1);the conversion of 1,5-gluconolactone to gluconic acid (Step 1a); theconversion of gluconic acid to 5-ketogluconate (5-KGA) (Step 14); theconversion of 5-ketogluconate (5-KGA) to 4,6-dihydroxy 2,5-diketohexanoate (2,5-DDH) (Step 16); the conversion of 4,6-dihydroxy2,5-diketo hexanoate (2,5-DDH) to 4-deoxy-5-threo-hexosulose uronate(DTHU) (Step 4); and the conversion of 4-deoxy-5-threo-hexosuloseuronate (DTHU) to DDG (Step 5).
 37. The method of claim 33 wherein thesubstrate is glucose and the product is DDG, comprising the steps of:the conversion of D-glucose to 1,5-gluconolactone (Step 1); theconversion of 1,5-gluconolactone to gluconic acid (Step 1a); theconversion of gluconic acid to 5-ketogluconate (5-KGA) (Step 14); theconversion of 5-ketogluconate (5-KGA) to L-Iduronic acid (Step 15); theconversion of L-Iduronic acid to 4-deoxy-5-threo-hexosulose uronate(DTHU) (Step 7B); the conversion of 4-deoxy-5-threo-hexosulose uronate(DTHU) to DDG (Step 5).
 38. The method of claim 33 wherein the substrateis glucose and the product is DDH, comprising the steps of: theconversion of D-glucose to 1,5-gluconolactone (Step 1); the conversionof 1,5-gluconolactone to guluronic acid lactone (Step 19); theconversion of guluronic acid lactone to guluronic acid (Step 1B); theconversion of guluronic acid to DEHU (Step 17A); the conversion of DEHUto DDH (Step 7A).
 39. The method of claim 33 wherein the substrate isglucose and the product is DDH, comprising the steps of: the conversionof D-glucose to 1,5-gluconolactone (Step 1); the conversion of1,5-gluconolactone to gluconic acid (Step 1a); the conversion ofgluconic acid to guluronic acid (Step 6); the conversion of guluronicacid to DEHU (Step 17A); the conversion of DEHU to DDH (Step 7A). 40.The method of claim 33 wherein the one or more conversion steps is theconversion of DTHU to DDG (Step-5).
 41. The method of claim 33 whereinthe one or more conversion steps is the conversion of gluconic acid toguluronic acid (Step-6).
 42. The method of claim 33 wherein the one ormore conversion steps is the conversion of DEHU to DDH (Step 7A). 43.The method of claim 33 wherein the one or more conversion steps is theconversion of guluronic acid to DEHU (Step 17A).
 44. The method of claim2 wherein the conversion of guluronic acid into D-glucarate is performedby a uronate dehydrogenase of SEQ ID NO: 1-3 or a homolog having atleast 70% sequence identity to SEQ ID NOs: 1-3; or by a uronatedehydrogenase encoded by a nucleic acid of SEQ ID NOs: 4-6 or a homologhaving at least 70% sequence identity to a nucleic acid of SEQ ID NOs:4-6. The method of claim 4 wherein the conversion of L-iduronic acidinto Idaric acid is performed by a uronate dehydrogenase of SEQ ID NO:1-3 or a homolog having at least 70% sequence identity to SEQ ID NOs:1-3; or by a uronate dehydrogenase encoded by a nucleic acid of SEQ IDNOs: 4-6 or a homolog having at least 70% sequence identity to a nucleicacid of SEQ ID NOs: 4-6.
 45. The method of claim 3 wherein theconversion of 5-KGA into L-iduronic acid is performed by an isomerase ofSEQ ID NOs: 7-19 or a homolog having at least 70% sequence identity toan isomerase of SEQ ID NOs: 7-19; or by an isomerase encoded by anucleic acid of SEQ ID NOs: 20-32 or a homolog having at least 70%sequence identity to a nucleic acid of SEQ ID NOs: 20-32.
 46. The methodof claim 5 wherein the conversion of 5-KGA into 2,5-DDH is performed bya gluconate dehydratase of SEQ ID NOs: 33-35 or a homolog having atleast 70% sequence identity to a gluconate dehydratase of SEQ ID NOs:33-35; or by a gluconate dehydratase encoded by a nucleic acid of SEQ IDNOs: 36-38 or a homolog having at least 70% sequence identity to anucleic acid of SEQ ID NOs: 36-38.
 47. The method of claim 6 wherein theconversion of 1,5-gluconolactone into gulurono-lactone is performed byan alditol oxidase of SEQ ID NOs: 39-46 or a homolog having at least 70%sequence identity to an alditol oxidase of SEQ ID NOs: 39-46; or by analditol oxidase encoded by a nucleic acid of SEQ ID NOs: 47-54 or ahomolog having at least 70% sequence identity to a nucleic acid of SEQID NOs: 47-54.